Methods and compositions for immunomodulation

ABSTRACT

Provided are cells containing exogenous antigen and uses thereof.

RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 15/301,046,filed Sep. 30, 2016, which is a U.S. National Stage Application under 35U.S.C. § 371 of International Application No. PCT/US2015/020614, filedMar. 13, 2015, which claims priority under 35 U.S.C. § 119 to U.S.Provisional Application No. 61/973,764, filed Apr. 1, 2014, U.S.Provisional Application No. 61/973,763, filed Apr. 1, 2014, U.S.Provisional Application No. 61/991,319, filed May 9, 2014, U.S.Provisional Application No. 62/006,825, filed Jun. 2, 2014, U.S.Provisional Application No. 62/006,828, filed Jun. 2, 2014, U.S.Provisional Application No. 62/006,829, filed Jun. 2, 2014, U.S.Provisional Application No. 62/006,832, filed Jun. 2, 2014, U.S.Provisional Application No. 62/025,367, filed Jul. 16, 2014, U.S.Provisional Application No. 62/059,100, filed Oct. 2, 2014, andInternational Application No. PCT/US2014/065304, filed Nov. 12, 2014.The entire contents of each of the foregoing applications areincorporated herein by reference.

FIELD OF THE INVENTION

The field of the invention is pharmaceutical compositions for thetreatment of diseases and disorders.

BACKGROUND OF THE INVENTION

Aberrant immune activation is a hallmark of many human diseases andconditions. Autoimmune diseases arise when the body's immune systemimproperly senses an autologous antigen as non-self and attacks thebody's own tissues. Inflammatory diseases and allergies can arise whenthe body's immune system is improperly triggered by common food-borne orenvironmental antigens. Polypeptides and proteins used to treat a rangeof human diseases are often destroyed, neutralized, or otherwiserendered ineffective by immune cells that respond to them as though theywere foreign antigens.

Current treatment of diseases of improper immune activation involvesimmunosuppression with chemical agents like corticosteroids, orinhibitors of inflammatory mediators like anti-histamines, antibodies,or cytokines. These generalized treatments are associated withsignificant morbidities, such as susceptibility to infection, becausethey broadly suppress the immune system.

For some severe allergies, clinical testing is underway to induce“tolerance” to allergens by exposure to slowly increasing doses of theoffending protein over time. To date theses treatments lack long-termefficacy and are associated with a risk of severe anaphylaxis.

There is a need for novel therapeutics to treat these diseases.

SUMMARY OF THE INVENTION

The invention, in certain aspects, relates to isolated enucleatedhematopoietic cells expressing an antigen. Enucleated hematopoieticcells will be referred to herein as “EHCs” (or in its singular form:“EHC”). In some embodiments, the enucleated hematopoietic cells or EHCslack nuclear material. For example, the EHCs can be are erythroid cellsor thromboid cells. In some embodiments, EHCs lacking nuclear materialare red blood cells, erythrocytes, reticulocytes, or platelets. In someembodiments, the enucleated hematopoietic cells or EHCs are nucleatedprecursor erythroid cells or precursor thromboid cells that are, e.g.,induced to lose their nuclear material or are rendered functionallyenucleated and incapable of replication. In some embodiments, theexogenous antigen-expressing EHC is a circulating cell, such as a redblood cell. In some embodiments, the exogenous antigen-expressing EHC iscultured from a hematopoietic precursor using defined factors. In someembodiments, the exogenous antigen-expressing EHC is a thromboid cell,such as a platelet. In some embodiments the thromboid cell is culturedfrom a hematopoietic precursor using defined factors. In someembodiments, the exogenous antigen-expressing EHC is a primary cellisolated from a patient, for either autologous or allogeneic use, thatis contacted with an antigen.

Certain aspects of the invention relate to exogenous antigen-expressingEHCs that are capable of inducing immune tolerance when administered toa subject, e.g. in form of a pharmaceutical composition comprising theexogenous antigen-expressing EHCs. The exogenous antigen expressed bythe EHCs can be tailored to a specific disease, disorder or condition.The exogenous antigen-expressing EHCs can comprise antigen in multipleways, such as e.g. surface display, intracellular expression,intracellular loading, or surface conjugation, of the antigen ofinterest. The exogenous antigen-expressing EHCs may manage diseases ofaberrant immune activation more effectively and/or with fewer sideeffects than existing treatments. For example, exogenousantigen-expressing EHCs may selectively modulate the immune system whileleaving the broader immune system physiology substantially unperturbed.In some embodiments, exogenous antigen-expressing EHCs may induce thedestruction, deactivation, and/or anergy of antigen-specific T and Blymphocytes. Alternatively or in addition, exogenous antigen-expressingEHCs may induce the proliferation of antigen-specific regulatory Tlymphocytes.

Certain aspects of the invention relate to exogenous antigen-expressingEHCs that comprise exogenous antigen that is recognized by immune cellsin autoimmune diseases, such as, e.g. multiple sclerosis, type 1diabetes, rheumatoid arthritis, and membranous nephritis.

Certain aspects of the invention relate to exogenous antigen-expressingEHCs that comprise exogenous antigen that is recognized by immune cellsin inflammatory diseases, such as, e.g. Crohn's disease, ulcerativecolitis, celiac disease, or other idiopathic inflammatory bowl diseases.

Certain aspects of the invention relate to exogenous antigen-expressingEHCs that comprise exogenous antigen that is recognized by immune cellsin human leukocyte antigen (HLA) mismatch-mediated diseases, such as,e.g. graft-versus-host disease or organ transplant rejection.

Certain aspects of the invention relate to exogenous antigen-expressingEHCs that comprise exogenous antigen that is recognized by immune cellsin allergic diseases, such as, e.g. asthma, peanut allergy, shellfishallergy, pollen allergy, milk protein allergy, insect sting allergy, andlatex allergy.

Certain aspects of the invention relate to exogenous antigen-expressingEHCs that comprise exogenous antigen that is a therapeutic protein whoseefficacy or potency is reduced or impaired by immune cells, such as,e.g., clotting factor VIII in hemophilia A, clotting factor IX inhemophilia B, anti-tumor necrosis factor alpha (TNFα) antibodies inrheumatoid arthritis and other inflammatory diseases, glucocerebrosidasein Gaucher's disease, or asparaginase in acute lymphoblastic leukemia(ALL).

Certain aspects of the invention relate to exogenous antigen-expressingEHCs that comprise exogenous antigen that comprises full-length,truncations, and chimeric fusions of polypeptides that a) mediatecomplement regulation, b) that mediate binding and sequestration ofimmune complexes, c) autoimmune antibodies, or d) pathogenic particles.In some embodiments, the exogenous antigen comprises full-length,truncations, and chimeric fusions of polypeptides that are enzymaticallyactive in the conversion of one small molecule substrate into anothersmall molecule product or of one polypetide substrate into a secondpolypeptide product, including, e.g., cleavage of the polypeptidesubstrate.

The invention, in certain aspects, also provides methods of treatment ofdisease using the exogenous antigen-expressing EHCs and pharmaceuticalcompositions thereof provided herein.

In some aspects, disclosed herein is a method of inducing immunetolerance. The method comprises administering to a human subjectsuffering from or at risk of developing an autoimmune disease, disorderor condition, a pharmaceutical composition comprising an enucleatedhematopoietic cell expressing an exogenous antigen, wherein thepharmaceutical composition is administered in an amount effective toinduce immune tolerance in the subject to the antigen mediating theautoimmune disease, disorder or condition.

In some embodiments, the autoimmune disease is selected from the groupconsisting of multiple sclerosis, type 1 diabetes, and those listed inTable F.

In some embodiments, the method further comprises administering thepharmaceutical composition at least twice over a treatment period suchthat the autoimmune disease, disorder or condition is treated, or asymptom thereof is decreased.

In certain embodiments, the method further comprises administering thepharmaceutical composition at least twice over a treatment period suchthat the autoimmune disease, disorder or condition is prevented.

In other embodiments, the method further comprises administering thepharmaceutical composition a sufficient number of times over a treatmentperiod such that the percentage of antigen-specific immune cells issubstantially decreased during the treatment period.

In some embodiments, the immune cell is a T cell. In some embodiments,the immune cell is a B cell.

In some embodiments, the decrease in concentration of antigen-specificimmune cells is measured by flow-cytometry from a biological sampletaken from the subject.

In some embodiments, the biological sample is a lymph node biopsy, aspleen sample, or peripheral blood.

In some embodiments, the concentration of antigen-specific immune cellsis decreased by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, 99.5%, 99.9%, 99.99%, or greater than 99.99% during part orthe entirety of the treatment period.

In other embodiments, the concentration of antigen-specific immune cellsis decreased by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, 99.5%, 99.9%, 99.99%, or greater than 99.99% within about 1,5, 10, 15, 20, 30, 40, or 50 minutes, or about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours, or1, 2, 3, 4, 5, or 6 days or about 1, 2, 3, 4, 5, or 6 weeks of theadministration.

In some embodiments, the pharmaceutical composition is administered asufficient number of times over a treatment period such that theconcentration of antigen-specific immune cells is substantiallydecreased for at least about one week, two weeks, three weeks, fourweeks, one month, two months, three months, four months, five months,six months, or greater than six months.

In certain embodiments, the pharmaceutical composition is administered asufficient number of times over a treatment period such that theconcentration of antigen-specific immune cells is substantiallydecreased for a period of time at least as long as the treatment period.

In some embodiments, the pharmaceutical composition is administered asufficient number of times over a treatment period such that theconcentration of antigen-specific antibodies in circulation issubstantially decreased during the treatment period.

In some embodiments, the concentration of antigen-specific antibodies incirculation is measured by ELISA.

In some embodiments, the concentration of antigen-specific circulatingantibody is decreased by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or greater than 99.99% during partor the entirety of the treatment period.

In some embodiments, the concentration of antigen-specific antibody isdecreased by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, 99.5%, 99.9%, 99.99%, or greater than 99.99% within about 1, 5, 10,15, 20, 30, 40, or 50 minutes, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours, or 1, 2, 3,4, 5, or 6 days or about 1, 2, 3, 4, 5, or 6 weeks of theadministration.

In certain embodiments, the pharmaceutical composition is administered asufficient number of times over a treatment period such that theconcentration of antigen-specific circulating antibody is substantiallydecreased for at least about one week, two weeks, three weeks, fourweeks, one month, two months, three months, four months, five months,six months, or greater than six months.

In certain embodiments, the pharmaceutical composition is administered asufficient number of times over a treatment period such that theconcentration of antigen-specific circulating antibody is substantiallydecreased for a period of time at least as long as the treatment period.

In some embodiments, the pharmaceutical composition is administered asufficient number of times over a treatment period such that thepercentage of antigen-specific regulatory T cells is substantiallyincreased during the treatment period.

In some embodiments, the decrease in concentration of antigen-specificimmune cells is measured by flow-cytometry from a biological sampletaken from the subject.

In some embodiments, the biological sample is a lymph node biopsy, aspleen sample, or peripheral blood.

In certain embodiments, the concentration of antigen-specific regulatoryT cells is increased by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or greater than 99.99% during partor the entirety of the treatment period.

In certain embodiments, the concentration of antigen-specific regulatoryT cells is increased by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or greater than 99.99% within about1, 5, 10, 15, 20, 30, 40, or 50 minutes, or about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours,or 1, 2, 3, 4, 5, or 6 days or about 1, 2, 3, 4, 5, or 6 weeks of theadministration.

In some embodiments, the pharmaceutical composition is administered asufficient number of times over a treatment period such that theconcentration of antigen-specific regulatory T cells is substantiallyincreased for at least about one week, two weeks, three weeks, fourweeks, one month, two months, three months, four months, five months,six months, or greater than six months.

In some embodiments, the pharmaceutical composition is administered asufficient number of times over a treatment period such that theconcentration of antigen-specific regulatory T cells is substantiallyincreased for a period of time at least as long as the treatment period.

In some embodiments, the pharmaceutical composition is administered asufficient number of times over a treatment period such that one or moresymptoms of the autoimmune disease, disorder or condition is prevented,decreased or delayed.

In some embodiments, the treatment period is not longer than a year, sixmonths, three months, two months, one month, two weeks, one week, threedays, two days, one day.

In certain embodiments, the time interval between administrations withinthe treatment period is no longer than the period in which the number ofenucleated hematopoietic cells expressing an exogenous antigen isreduced to less than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the number ofenucleated hematopoietic cells expressing an exogenous antigen presentin the administered pharmaceutical composition.

In some embodiments, the frequency of administration is sufficient toeffectively reduce the concentration of antigen-specific immune cellsbelow a level that is associated with a symptom of autoimmune disease,disorder or condition.

In certain embodiments, the frequency of administration is sufficient toeffectively reduce the concentration of antigen-specific circulatingantibody below a level that is associated with a symptom of autoimmunedisease, disorder or condition.

In ceratin embodiments, the frequency of administration is sufficient toeffectively increase the concentration of antigen-specific, regulatory Tcells above a threshold level that is associated with a symptom ofautoimmune disease, disorder or condition.

In some embodiments, the enucleated hematopoietic cell is an erythroidcell, a thromboid cell, or a precursor thereof. In some embodiments, theerythroid cell is an erythrocyte or a reticulocyte. In some embodiments,the thromboid cell is a platelet.

In some embodiments, the enucleated hematopoietic cell is isolated froma donor. In some embodiments, the enucleated hematopoietic cell isautologously derived from the subject. In some embodiments, theenucleated hematopoietic cell is allogeneically derived. In someembodiments, the enucleated hematopoietic cell is xenogeneicallyderived.

In some embodiments, the nucleated hematopoietic cell is derived from anucleated precursor cell by a culture-based process that induces theexpulsion of its nucleus. In some embodiments, the enucleatedhematopoietic cell is generated from a nucleated precursor cell that ischemically or physical manipulated to remove its nucleus.

In some embodiments, the enucleated hematopoietic cell is generated byirradiation or chemical destruction of the nucleus of a nucleatedprecursor cell. In some embodiments, the chemical destruction is carriedout with Cytochalasin B. In some embodiments, the irradiation is carriedout with at least 5 Gy, 7 Gy, 10 Gy, 15 Gy, 25 Gy, 30 Gy, 40 Gy or atleast 50 Gy.

In some embodiments, the exogenous antigen is a polypeptide encoded byan exogenous nucleic acid.

In some embodiments, the exogenous antigen is associated with the cellmembrane of the enucleated hematopoietic cell.

In some embodiments, the exogenous antigen is a fusion or a chimerapolypeptide.

In some embodiments, the fusion or chimera comprises at least one of anS domain, an A domain or a U domain, wherein the S domain is a surfacedomain on the enucleated hematopoietic cell, wherein the A domain is ananchor in or on the cell membrane, wherein the U domain faces theintracellular, unexposed side of the enucleated hematopoietic cell, andwherein the S domain, the A domain, and/or the U domain are of differentpolypeptide origin.

In some embodiments, the S domain and/or the A domain comprises at least5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150,200, 250, or at least 500 amino acids. In some embodiments, the S domainand/or A domain comprises at least 500, 750, or at least 1,000 aminoacids.

In some embodiments, the exogenous antigen is selected from the groupconsisting of myelin basic protein, proteolipid protein, myelinoligodendrocyte glycoprotein, pancreatic beta cell antigen, insulin, andthose listed in Table F, Table 6, and Table 8.

In some embodiments, the enucleated hematopoietic cell comprises atleast 10 copies, 100 copies, 1,000 copies, 10,000 copies, 25,000 copies,50,000 copies, 100,000 copies, 500,000 copies, 1,000,000 copies, or2,000,000 copies of the exogenous antigen.

In some embodiments, the pharmaceutical composition further comprises apharmaceutically active agent.

In some embodiments, the method further comprises the step ofadministering a pharmaceutically active agent, wherein thepharmaceutically active agent is administered prior to, after, orconcurrent with the pharmaceutical composition.

In some embodiments, the pharmaceutical composition is administeredintravenously.

In some embodiments, the pharmaceutically active agent is selected froma biological agent, a small molecule agent, or a nucleic acid agent.

In some embodiments, the pharmaceutical composition further comprises apharmaceutically acceptable carrier.

In certain embodiments, the method further comprises the step ofselecting for treatment a subject suffering from or at risk of anautoimmune disease, disorder or condition selected from the groupconsisting of: thrombotic thrombocytopenic purpura, CAPS, APS,myasthenia gravis, Goodpasture's syndrome, membraneous nephritis, type 1diabetes, rheumatoid arthritis, multiple sclerosis, Crohn's disease, orthose listed in Table F and Table G.

In some aspects, disclosed herein is a pharmaceutical compositioncomprising an enucleated hematopoietic cell expressing an exogenousantigen, wherein administration of an effective amount of thepharmaceutical composition is capable of inducing immune tolerance in ahuman subject suffering from or at risk of developing an autoimmunedisease, disorder or condition administered by the method of any one ofthe preceding claims.

In some embodiments, the pharmaceutical composition further comprises apharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition comprises apopulation of hematopoietic cells expressing an exogenous antigen. Insome embodiments, the pharmaceutical composition comprises at least1x103 hematopoietic cells expressing an exogenous antigen.

In certain embodiments of the pharmaceutical composition, thehematopoietic cells expressing an exogenous antigen are provided in avolume of about 10 nl, 100 nl, 1 μl, 10 μl, 100 μl, 1 ml, 10 ml, 20 ml,or 50 ml. In other embodiments of the pharmaceutical composition, thehematopoietic cells expressing an exogenous antigen are provided in avolume of about 1 ml, 10 ml, 20 ml, 50 ml, 100 ml, 250 ml, or 500 ml.

In some embodiments of the pharmaceutical composition, the compositionis formulated for long-term storage. In some embodiments of thepharmaceutical composition, the composition is frozen. In someembodiments, the pharmaceutical composition comprises a pharmaceuticallyactive agent.

In certain embodiments of the pharmaceutical composition, thepharmaceutically active agent is selected from a biological agent, asmall molecule agent, or a nucleic acid agent.

In some aspects, disclosed herein is a dosage form comprising thecompositions described herein formulated as a liquid suspension forintravenous injection.

In some aspects, disclosed herein is a medical device comprising acontainer holding the pharmaceutical compositions described herein andan applicator for intravenous injection of the pharmaceuticalcomposition to the subject.

In some aspects, disclosed herein is a medical kit comprising thepharmaceutical compositions described herein and a medical device forintravenous injection of the pharmaceutical composition to the subject.

In some aspects, disclosed herein are hematopoietic cells expressing anexogenous antigen of the pharmaceutical composition administered by anyof the methods described herein.

In some aspects, disclosed herein is a population of hematopoietic cellsexpressing an exogenous antigen as disclosed herein.

In some embodiments, the population of hematopoietic cells expressing anexogenous antigen is formulated as a liquid.

In some embodiments, the population of hematopoietic cells expressing anexogenous antigen is frozen.

In some aspects, disclosed herein is an isolated antigen expressed bythe hematopoietic cell population described herein.

In some aspects, disclosed herein is an exogenous nucleic acid encodingthe exogenous antigen described herein.

In some aspects, disclosed herein is an enucleated hematopoietic cellcomprising an exogenous antigen that comprises at least one of an Sdomain, an A domain or a U domain, wherein the S domain is anextracellular surface domain, the A domain is an anchor, and the Udomain is intracellularly localized, and wherein the enucleatedhematopoietic cell is capable of inducing immune tolerance whenadministered to a subject.

In some embodiments of the enucleated hematopoietic cell disclosedherein, the exogenous antigen is a fusion or a chimeric polypeptide.

In some embodiments of the enucleated hematopoietic cell disclosedherein, the S domain, the A domain, and/or the U domain are of differentpolypeptide origin.

In certain embodiments of the enucleated hematopoietic cell disclosedherein, the S domain and/or the A domain comprises at least 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or atleast 500 amino acids.

In certain embodiments of the enucleated hematopoietic cell disclosedherein, the S domain and/or A domain comprises at least 500, 750, or atleast 1,000 amino acids.

In some embodiments of the enucleated hematopoietic cell disclosedherein, the exogenous antigen is selected from the group consisting ofmyelin basic protein, proteolipid protein, myelin oligodendrocyteglycoprotein, pancreatic beta cell antigen, insulin, and those listed inTable F, Table 6, and Table 8.

In some embodiments, the enucleated hematopoietic cell comprises atleast 10 copies, 100 copies, 1,000 copies, 10,000 copies, 25,000 copies,50,000 copies, 100,000 copies, 500,000 copies, 1,000,000 copies, or2,000,000 copies of the exogenous antigen.

In certain embodiments, the enucleated hematopoietic cell is anerythroid cell, a thromboid cell, or a precursor thereof. In someembodiments, the erythroid cell is an erythrocyte or a reticulocyte. Insome embodiments, the thromboid cell is a platelet.

In some embodiments, the enucleated hematopoietic cell is isolated froma donor. In some embodiments, the enucleated hematopoietic cell isautologously derived from the subject. In some embodiments, theenucleated hematopoietic cell is allogeneically derived. In someembodiments, the enucleated hematopoietic cell is xenogeneicallyderived.

In certain embodiments, the enucleated hematopoietic cell is derivedfrom a nucleated precursor cell by a culture-based process that inducesthe expulsion of its nucleus.

In certain embodiments, the enucleated hematopoietic cell is generatedfrom a nucleated precursor cell that is chemically or physicalmanipulated to remove its nucleus.

In certain embodiments, the enucleated hematopoietic cell is generatedby irradiation or chemical destruction of the nucleus of a nucleatedprecursor cell. In some embodiments, the chemical destruction is carriedout with Cytochalasin B. In some embodiments, the irradiation is carriedout with at least 5 Gy, 7 Gy, 10 Gy, 15 Gy, 25 Gy, 30 Gy, 40 Gy or atleast 50 Gy.

In some embodiments of the enucleated hematopoietic cell disclosedherein, the exogenous antigen is a polypeptide encoded by an exogenousnucleic acid.

In some embodiments of the enucleated hematopoietic cell disclosedherein, the cell is derived from a human source.

In some embodiments of the enucleated hematopoietic cell disclosedherein, the cell is derived from a non-human source, selected from thegroup consisting of pig, chimpanzee, macaque, a non-human primate, and anon-primate mammal.

In some embodiments of the enucleated hematopoietic cell disclosedherein, the polypeptide antigen is localized intracellularly. In someembodiments of the enucleated hematopoietic cell disclosed herein, thepolypeptide antigen is localized extracellularly on the surface of thecell. In some embodiments of the enucleated hematopoietic cell disclosedherein, the polypeptide antigen is fused to an endogenous cell protein.In some embodiments of the enucleated hematopoietic cell disclosedherein, the polypeptide antigen is fused to an intracellular region ofan endogenous transmembrane protein. In some embodiments of theenucleated hematopoietic cell disclosed herein, the polypeptide antigenis fused to an extracellular region of an endogenous transmembraneprotein. In some embodiments of the enucleated hematopoietic celldisclosed herein, the polypeptide antigen is fused to aglycosylphosphatidylinisotol (GPI) anchored protein.

In some aspects, disclosed herein is a tissue culture batch comprisingthe enucleated hematopoietic cells described herein.

In some aspects, disclosed herein is a population of the enucleatedhematopoietic cells described herein.

In some aspects, disclosed herein is a pharmaceutical compositioncomprising the cell populations described herein.

In some aspects, disclosed herein is a method of inducing immunetolerance comprising administering to a subject in need thereof thepharmaceutical compositions described herein in an amount and/or afrequency sufficient to induce immune tolerance in the subject.

In some aspects, disclosed herein is a method of treating an immuneactivation disease comprising administering to a subject in need thereofthe pharmaceutical compositions described herein in an amount and/orfrequency sufficient to treat the immune activation disease.

In some embodiments, the disease is selected from the group consistingof a self-antibody mediated disease, an autoimmune disease, aninflammatory disease, an allergic disease, an HLA-mismatch mediateddisease, and a disease treatable by an immunogenic therapeutic protein.

In some aspects, disclosed herein is a method of reducing or alleviatingan immune activation in response to a therapeutic protein treatmentregimen comprising administering to a subject in need thereof thepharmaceutical compositions described herein in an amount and/orfrequency sufficient to substantially reduce or alleviate the immuneactivation.

In some embodiments, the therapeutic protein is selected from the groupconsisting of those listed in Table I, Table J, and Table 7.

In some aspects, disclosed herein is an expression vector comprising anucleic acid sequence encoding an endogenous erythroid cell proteinfused with one or more exogenous polypeptide antigens selected from thegroup consisting of those listed in Table F, Table G, Table H, Table I,Table J, Table 6, Table 7, and Table 8.

In some aspects, disclosed herein is a messenger RNA comprising anucleic acid sequence encoding an endogenous erythroid cell proteinfused with one or more exogenous polypeptide antigens selected from thegroup consisting of those listed in Table F, Table G, Table H, Table I,Table J, Table 6, Table 7, and Table 8.

In some aspects, disclosed herein is a method of inducing immunetolerance comprising administering to a human subject suffering from orat risk of developing an allergen-mediated disease, disorder orcondition, a pharmaceutical composition comprising an enucleatedhematopoietic cell expressing an exogenous antigen, wherein thepharmaceutical composition is administered in an amount effective toinduce immune tolerance in the subject to the allergen mediating thedisease, disorder or condition.

In certain embodiments, the exogenous antigen is selected from the groupconsisting of Ara h2, 2S albumin, hyalauronidase, and those listed inTable H.

In certain embodiments, the allergen-mediated disease, disorder orcondition is selected from the group consisting of peanut allergy, treenut allergy, insect venom allergy, and those listed in Table H.

In some aspects, disclosed herein is a method of inducing immunetolerance comprising administering to a human subject suffering from orat risk of developing a human leukocyte antigen (HLA) mismatch-mediateddisease, disorder or condition, a pharmaceutical composition comprisingan enucleated hematopoietic cell expressing an exogenous antigen,wherein the pharmaceutical composition is administered in an amounteffective to induce immune tolerance in the subject to the HLA mediatingthe disease, disorder or condition.

In some aspects, disclosed herein is a method of inducing immunetolerance comprising: administering to a human subject suffering from orat risk of developing a disease, disorder or condition that can betreated by an immunogenic therapeutic molecule, a pharmaceuticalcomposition comprising an enucleated hematopoietic cell expressing anexogenous antigen, wherein the pharmaceutical composition isadministered in an amount effective to induce immune tolerance in thesubject to the immunogenic therapeutic molecule used to treat thedisease, disorder or condition.

In some embodiments, the therapeutic molecule is selected from the groupconsisting of Recombinant (factor VIII), Benefix (factor IX), Humira(anti-TNFα), and those listed in Table I, Table J, and Table 7.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a collection of flow cytometry plots of red blood cellscontacted with fluorescently labeled IgG encapsulated within liposomes.Cells are shown that are incubated with no liposomes (left), a low doseof liposomes (center), and a high dose of liposomes (left). On thebottom histograms, the percentage of cells that are fluorescent isshown.

FIG. 2 is a plot of cell surface expression levels assessed byquantitative flow cytometry. The plot shows of various cell surfacereceptors—glycophorin A (solid triangles), cKIT (dashed squares),transferrin receptor (dotted diamonds)—and an exogenous surfacetransgene (open circles) during the course of erythroid celldifferentiation.

FIG. 3A-C, F, I-M, O-Z, and AA-AU is a collection of flow cytometryplots and Western blots that demonstrate the expression of a vast arrayof exemplary antigens on the surface, in the cytoplasm, as fusions, andas intact proteins, in three cell types, enucleated erythroid cells,nucleated erythroid precursor cells, and erythroleukemic cells.

FIG. 3A-C, F, I-M, and O-S shows the exogenous expression of surface andcytoplasmic proteins on enucleated cultured erythroid cells.

FIG. 3A—Expression of glycophorin A with an HA epitope tag at thecytoplasmic C terminus assessed by expression of co-translated GFP.

FIG. 3B—Expression of glycophorin A with an HA epitope tag at the Nterminus between the leader sequence and the body of the gene assessedby anti-HA staining.

FIG. 3C—Expression of complement receptor 1-derived fragment of ˜70 kDawith an HA epitope tag at the N terminus assessed by anti-HA staining.

FIG. 3F—Expression of antibody scFv as N terminal fusion to glycophorinA assessed by anti-HA staining.

FIG. 3I—Expression of antibody scFv fused to C terminus of Kell-derivedfragment of 71 amino acids assessed by anti-HA staining.

FIG. 3J—Expression of antibody scFv fused to C terminus of Kell-derivedfragment of 79 amino acids assessed by anti-HA staining.

FIG. 3K—Expression of CD55 with HA epitope tag at the extracellular Nterminus after the leader sequence assessed by anti-HA staining.

FIG. 3L—Expression of CD59 with HA epitope tag at the extracellular Nterminus after the leader sequences assessed by anti-HA staining.

FIG. 3M—Expression of antibody scFv fused to N-terminus of CD55-derivedfragment of 37 amino acids, assessed by anti-HA Western blot.

FIG. 3O—Cytoplasmic expression of adenosine deaminase fused to HA tagassessed by anti-HA Western blot. Expected size approximately 40 kDa.

FIG. 3P—Cytoplasmic expression of phenylalanine hydroxylase fused to HAtag assessed by anti-HA Western blot. Expected size approximately 33kDa.

FIG. 3Q—Cytoplasmic expression of phenylalanine hydroxylase fused toadenosine deaminase and an HA tag assessed by anti-HA Western blot.

FIG. 3R—Cytoplasmic expression of adenosine deaminase fused to theintracellular C terminus of glycophorin A assessed by anti-HA Westernblot. Expected size approximately 55 kDa.

FIG. 3S—Cytoplasmic expression of phenylalanine hydroxylase fused to theintracellular C terminus of glycophorin A assessed by anti-HA Westernblot. Expected size approximately 50 kDa.

FIG. 3T-AO shows the exogenous expression of surface and cytoplasmicproteins on nucleated cultured erythroid precursor cells.

FIG. 3T—Expression of glycophorin A with an HA epitope tag at thecytoplasmic C terminus assessed by expression of co-translated GFP.

FIG. 3U—Expression of glycophorin A with an HA epitope tag at the Nterminus between the leader sequence and the body of the gene assessedby anti-HA staining.

FIG. 3V—Overexpression of complement receptor 1 assessed by anti-CR1staining.

FIG. 3W—Expression of complement receptor 1-derived fragment of ˜70 kDawith an HA epitope tag at the N terminus assessed by anti-HA staining.

FIG. 3X—Expression of complement receptor 1-derived fragment of ˜210 kDawith an HA epitope tag at the N terminus assessed by anti-HA staining.

FIG. 3Y—Expression of complement receptor 1-derived fragment of ˜230 kDafused to the N terminus of glycophorin A with an HA epitope tag at the Nterminus assessed by anti-HA staining.

FIG. 3Z—Expression of antibody scFv as N terminal fusion to glycophorinA assessed by anti-HA staining.

FIG. 3AA—Expression of antibody scFv fused to the extracellular Cterminus of Kell, assessed by anti-HA staining. Expected sizeapproximately 108 kDa.

FIG. 3AB—Expression of HA tag fused to the extracellular C terminus ofKell, assessed by anti-HA staining.

FIG. 3AC—Expression of Kell-derived fragment of 71 amino acids with HAtag at the C (extracellular) terminus assessed by anti-HA staining.

FIG. 3AD—Expression of Kell-derived fragment of 79 amino acids with HAtag at the C terminus assessed by anti-HA staining.

FIG. 3AE—Expression of antibody scFv fused to C terminus of Kell-derivedfragment of 71 amino acids assessed by anti-HA staining.

FIG. 3AF—Expression of antibody scFv fused to C terminus of Kell-derivedfragment of 79 amino acids assessed by anti-HA staining.

FIG. 3AG—Expression of CD55 with HA epitope tag at the extracellular Nterminus after the leader sequence assessed by anti-HA staining

FIG. 3AH—Expression of CD59 with HA epitope tag at the extracellular Nterminus after the leader sequences assessed by anti-HA staining.

FIG. 3AI—Expression of antibody scFv fused to N-terminus of CD55-derivedfragment of 37 amino acids, assessed by anti-HA staining.

FIG. 3AJ—Expression of antibody scFv fused to N-terminus of CD59assessed by anti-HA staining.

FIG. 3AK—Cytoplasmic expression of adenosine deaminase fused to HA tagassessed by anti-HA Western blot. Expected size approximately 40 kDa.

FIG. 3AL—Cytoplasmic expression of phenylalanine hydroxylase fused to HAtag assessed by anti-HA Western blot. Expected size approximately 33kDa.

FIG. 3AM—Cytoplasmic expression of phenylalanine hydroxylase fused toadenosine deaminase and an HA tag assessed by flow cytometry forfluorescence from co-translated GFP.

FIG. 3AN—Cytoplasmic expression of adenosine deaminase fused to theintracellular C terminus of glycophorin A assessed by anti-HA Westernblot. Expected size approximately 55 kDa.

FIG. 3AO—Cytoplasmic expression of phenylalanine hydroxylase fused tothe intracellular C terminus of glycophorin A assessed by anti-HAWestern blot. Expected size approximately 50 kDa.

FIG. 3AP-AU shows the exogenous expression of surface and cytoplasmicproteins on K562 erythroleukemia cells.

FIG. 3AP—Overexpression of complement receptor 1 assessed by anti-CR1staining.

FIG. 3AQ—Expression of antibody scFv as N terminal fusion to glycophorinA assessed by anti-HA staining.

FIG. 3AR—Expression of antibody scFv fused to N-terminus of CD55-derivedfragment of 37 amino acids, assessed by anti-HA staining.

FIG. 3AS—Expression of antibody scFv fused to N-terminus of CD59assessed by anti-HA staining.

FIG. 3AT—Cytoplasmic expression of adenosine deaminase fused to HA tagassessed by anti-HA Western blot. Expected size approximately 40 kDa.

FIG. 3AU—Cytoplasmic expression of phenylalanine hydroxylase fused to HAtag assessed by anti-HA Western blot. Expected size approximately 33kDa.

FIGS. 4A-4C are a collection of flow cytometry histograms that measurefluorescence in primary platelets that have been transfected with mRNAencoding a fluorescent protein (GFP). (FIG. 4A) Untransfected platelets.(FIG. 4B) Platelets transfected with 3 ug GFP mRNA. (FIG. 4C) Plateletstransfected with 6.8 ug GFP mRNA.

FIGS. 5A-5D show protein expression and enzymatic activity of transgenicerythroid cells in culture. (FIG. 5A) is a Western blot of exogenouslyexpressed adenosine deaminase detected with an anti-HA antibody over thecourse of differentiation, from nucleated precursor cells (“Diff I D5”)through to enucleated erythroid cells (“Diff III D8”). (FIG. 5B) is abar chart of inosine produced from adenosine by intact adenosinedeaminase-expressing 293T cells. (FIG. 5C) is a Western blot of theexogenously expressed phenylalanine hydroxylase detected with an anti-HAantibody at various time points over the course of differentiation, fromnucleated precursor cells (“Diff I D5”) through to enucleated erythroidcells (“Diff III D8”). (FIG. 5D) is a bar chart of tyrosine producedfrom phenylalanine by lysates of cultured phenylalaninehydroxylase-expressing enucleated erythroid cells.

FIGS. 6A-6B show immune complex capture and transfer to macrophages bycultured erythroid cells that overexpress complement receptor 1 (CR1).(FIG. 6A) is a flow cytometry plot that shows the capture of fluorescentimmune complexes (white histogram) and complement-deficient immunecomplexes (shaded histogram) by cultured erythroid cells thatoverexpress CR1. (FIG. 6B) is a bar chart of flow cytometry dataassessing the uptake of fluorescent immune complexes (hashed bars),complement deficient immune complexes (gray bars), or no immunecomplexes (black bars) by macrophages (left set) or macrophagesincubated with cultured erythroid cells that overexpress CR1 (rightset).

FIGS. 7A-7D show the activity of an antibody scFv that binds hepatitis Bsurface antigen (scFv) on the surface of a cultured erythroid cell.(FIG. 7A) is a flow cytometry histogram showing binding of 450 nMantigen (white histogram) or no antigen (gray histogram). (FIG. 7B) is atitration of binding signal assessed by flow cytometry for a range ofantigen concentrations. (FIGS. 7C-D) are flow cytometry plots of bloodcells from mice that had been injected with fluorescent antigen andcultured erythroid cells that (FIG. 7C) do not or (FIG. 7D) do expressscFv. The y-axis measures antigen fluorescence. The x-axis measuresfluorescence of the cultured cells.

FIGS. 8A-8D show the specific clearance of circulating antibodiesmediated by exogenous antigen-expressing EHCs in vivo. (FIG. 8A) is aset of flow cytometry plots that show no binding (top) and binding(bottom) of circulating Dylight650-labeled mouse anti-HA antibody toCFSE-labeled cultured human erythroid cells isolated from a recipientmouse that either do not (top) or do (bottom) express HA epitope tag ontheir surface. The x-axis measures CFSE fluorescence. The y-axismeasures anti-HA antibody Dylight650 fluorescence. (FIG. 8B) is datafrom an HA epitope tag substrate ELISA comparing anti-HA antibody levelsover time in plasma collected from mice injected with anti-HA antibody(open circles, solid line), anti-HA antibody followed by cultured humanerythroid cells that do not express HA epitope tag (dashed line), oranti-HA antibody followed by cultured human erythroid cells that doexpress HA epitope tag (dotted line). (FIG. 8C) is a set of flowcytometry plots that show no binding (top) and binding (bottom) ofDylight650-labeled mouse anti-biotin antibody to CFSE-labeled primaryhuman erythrocytes that either are not (top) or are (bottom) conjugatedwith biotin on their surface. The x-axis measures CFSE fluorescence. They-axis measures anti-biotin antibody Dylight650 fluorescence. (FIG. 8D)is data from a biotin substrate ELISA comparing anti-biotin antibodylevels over time in plasma collected from mice injected with anti-biotinantibody (open circles, solid line), anti-biotin antibody followed bycultured human erythroid cells that are not conjugated to biotin (dashedline), or anti-biotin antibody followed by cultured human erythroidcells that are conjugated to biotin (dotted line).

FIGS. 9A-9B show the clearance rate of cultured human eyrthroid cells ina mouse. (FIG. 9A) is a representative flow cytometry dot-plot of drawnblood, stained for human glycophorin A (y-axis) and CFSE (x-axis), inwhich human cultured cells are double-positive. (FIG. 9B) is a plot ofthe clearance rate over time as a percentage of double-positive cellsremaining after NSG mice were injected with with human red blood cells(solid circles), cultured enucleated erythroid cells (dashed diamonds),cultured enucleated erythroid cells that express an intracellularexogenous protein (dotted squares) and cultured enucleated erythroidcells that express a surface exogenous protein (open triangles).

FIGS. 10A-10D are an assessment of adverse events following injection ofcultured human erythroid cells into a mouse. (FIGS. 10A-B) show levelsof (FIG. 10A) fibrinopeptide A and (FIG. 10B) fibrinopeptide B assessedby ELISA in plasma collected from mice 20 minutes (black), 6 hours(gray), and 48 hours (white) after injection with (1) human red bloodcells, (2) cultured human erythroid cells, (3) cultured human erythroidcells expressing an exogenous cytoplasmic protein, (4) cultured humanerythroid cells expressing an exogenous surface transgene, or (5)recombinant protein. (FIGS. 10C-D) show microscope images ofhistologically stained sections of spleen for mice injected with (

FIG. 10C) cultured human erythroid cells and (FIG. 10D) recombinantprotein.

FIGS. 11A-11B track the expression of exogenous protein on culturederythroid cells in circulation. (FIG. 11A) is flow cytometry data ofblood drawn from a mouse that was injected with cultured human erythroidcells expressing an exogenous surface protein, showing the the percentof cultured human erythroid cells that are HA-positive over time. (FIG.11B) is a Western blot of blood drawn from two mice, wherein one mousewas injected with cultured human erythroid cells expressing an exogenouscytoplasmic protein, and wherein the other mouse was injected with thepurified recombinantly-produced exogenous protein in the absence of anycells, showing the level of HA-containing protein in the blood overtime.

FIGS. 12A-12C are an assessment of expansion and differentiation ofcultured human erythroid cells. (FIG. 12A) is a plot of expansion ratesfor distinct cultures of in vitro differentiated erythroid cells thatcontain transgenes (dashed line and dotted line) and cells that do notcontain a transgene (solid line). (FIG. 12B) is a flow cytometry plot ofcell surface markers GPA and CKIT for distinct cultures of culturedhuman erythroid cells that do not (left) or do (right) contain atransgene. (FIG. 12C) is a flow cytometry plot of cultured humanerythroid cells that do not (left) or do (right) contain a transgene,wherein the cells are stained with DNA stain DRAQS (y-axis) andanti-glycophorin A (x-axis), which identifies distinct populations of(1) enucleated cells, (2) nucleated cells, and (3) nuclei.

FIG. 13A is a schematic of three ways in which an antigen may belocalized in an exogenous antigen-expressing EHC. FIG. 13B is aschematic of three ways in which an antigen localized in or on anexogenousan exogenous antigen-expressing EHC may act on a target incirculation. FIG. 13C is a schematic of an auto-catalytic fusion of anendogenous polypeptide anchor to an antigen utilizing aSpyTag-SpyCatcher mechanism.

DETAILED DESCRIPTION OF THE INVENTION

The invention, in certain aspects, provides isolated cells that areengineered or modified to contain exogenous antigens of interest. Incertain aspects, isolated EHCs of the invention comprise one or moreantigens that comprise or consist of polypeptides. In some embodiments,the antigen is a full-length protein. In some embodiments, the antigenis comprised of one or more polypeptides contained within thefull-length protein, of any length greater than approximately 7 aminoacids. The polypeptides comprising the antigen may comprise one or moreimmunological epitopes which may be conformational epitopes or may belinear epitopes. The antigen may be comprised of one or morepolypeptides from one or more different proteins. In certain aspects,EHCs of the invention comprise one or more antigens that comprise orconsist of carbohydrates. In certain aspects, EHCs of the inventioncomprise one or more antigens that comprise or consist of lipids. Incertain aspects, EHCs of the invention comprise one or more antigensthat comprise or consist of one or more polypeptides, lipids, and/orcarbohydrates, and any combination thereof. The cells can be circulatingcells, such as EHCs. The EHCs can be cultured from hematopoieticprecursors using defined factors such as e.g. stem cell factor,cytokines such as IL-3 and IL-6, insulin, transferrin, erythropoietin,hydrocortisone, and estrogens.

Aspects of the invention relate to methods of culturing EHCs to compriseexogenous antigens of interest. The exogenous antigens of interest canbe introduced by a number of methods, such as, e.g. intracellularexpression, cell-surface expression, fusion to an endogenous protein,conjugation by chemical or enzymatic means to a cell surface protein, orphysical loading into the intracellular space. The antigen-comprisingcells of the invention may be used as therapeutic agents.

Aspects of the invention relate to the use of these antigen-comprisingcells in the treatment of diseases of immune activation by the inductionof peripheral tolerance. In some aspects, induction of peripheraltolerance means the deletion or inactivation of antigen-specific immunecells, such as, e.g. CD8 T lymphocytes (CD8 T cells), CD4 T lymphocytes(CD4 T cells), CD4 T regulatory lymphocytes (Treg), or B lymphocytes (Bcells). Diseases of immune activation include autoimmune diseases, suchas, e.g. multiple sclerosis, type 1 diabetes, rheumatoid arthritis, andmembranous nephritis. Diseases of immune activation also includeinflammatory diseases, such as, e.g. Crohn's disease, ulcerativecolitis, celiac disease, or other idiopathic inflammatory bowl diseases.Diseases of immune activation also include allergic diseases, such as,e.g. asthma, peanut allergy, shellfish allergy, pollen allergy, milkprotein allergy, insect sting allergy, and latex allergy. Diseases ofimmune activation also include immune activation in response to atherapeutic protein, administered to treat a primary condition, thatlessens the efficacy of the therapeutic protein, such as, e.g., clottingfactor VIII in hemophilia A, clotting factor IX in hemophilia B,anti-tumor necrosis factor alpha (TNFa) antibodies in rheumatoidarthritis and other inflammatory diseases, glucocerebrosidase inGaucher's disease, or asparaginase in acute lymphoblastic leukemia(ALL).

Biology of Immune Tolerance

The body has evolved sophisticated mechanisms for the prevention ofaberrant immune activation and autoimmune disease, collectively termedimmune tolerance. Central tolerance refers to the antigen-specificdeletion of autoreactive T cells and B cells during development in theprimary lymphoid organs, e.g. thymus and bone marrow. Peripheraltolerance refers to the deletion or inactivation of mature T and Blymphocytes outside of the primary lymphoid organs. Peripheral toleranceincludes the suppression of autoreactive lymphocytes by regulatory Tcells (Tregs) or the induction of anergy or non-responsiveness inantigen-specific effector lymphocytes by exposure to continuous lowdoses of antigen in the absence of costimulatory “danger” signals. BothTreg activation and lymphocyte anergy can be induced by the secretion ofinhibitory factors such as, for example, TGF-beta, IL-10, and IL-4.

Immune activation in response to antigen often requires a secondary“danger” signal, such as a toll-like receptor ligand, often derived frommicrobial or viral pathogens (Matzinger, Annu Rev Immuno 1994). Suchdanger signals include double-stranded RNA, single-stranded DNA,lipopolysaccharide, bacterial lipoproteins, flagellin, zymosan, andothers. Antigen presenting cells that receive both antigen and dangersignal display costimulatory molecules on their surface, like CD80 andCD86, in addition to the antigenic peptides. T cells that recognize boththe antigenic peptide and the costimulatory molecules become activated.Those that receive just the antigenic peptide signal become anergic.

Therapeutic strategies that take advantage of antigen presentation inthe absence of danger signals to induce immune tolerance have beendeveloped for the experimental treatment of many food allergies. Thestudies take the form of prolonged exposure to increasing doses ofallergen with the intent to induce tolerance. Thirteen studies since2007 have tested a variety of common food allergens like peanut, milk,and egg, in this format. 50-100% of patients become sensitized, that is,able to survive a food challenge without anaphylaxis. However, long termtolerance is less successful, with only 25-50% of patients able totolerate antigen after one month off therapy. See, e.g. Burks et al.,New England Journal of Medicine 2012.

It is thought that allergy is IgE mediated, with activation of mastcells and basophils. Oral administration of the allergen in low dose,such as continuous feeding, induces Tregs via CD11c⁺ dendritic cellpresentation of antigen and secretion of TGF-beta, IL-10, and IL-4. Oraladministration at high doses induces antigen-specific T cell deletionand anergy via plasmacytoid dendritic cells. In human studies, oraladministration of allergen results in a decrease in IgE, mast cells, andbasophils, an increase in IgG4, TGF-beta, IL-10, and a temporary uptickin Tregs at the start of therapy. See, e.g. Herzog, Adv Drug Deliv Rev2013.

While not wishing to be limited to any particular mechanism, it isbelieved that peripheral immunologial tolerance can be induced by selfantigens from apoptosing cells (Griffith and Ferguson, Immunity 2011;Green et al., Nat Rev Immunol 2009). Though the exact mechanisms are notfully understood from a molecular perspective, self proteins such asHSP90 and other damage-associated molecular patterns facilitate uptakeby dendritic cells. Dendritic cell receptors like CD205 recognize thesesignals, cross-present antigen, and induce tolerogenic cytokines andsuppress costimulatory protein expression (Bonifaz, J Exp Med 2002).

Therapeutic strategies that harness the tolerogenic potential ofapoptosing cells to induce peripheral immune tolerance are underinvestigation. These strategies typically involve the chemical couplingof antigens of interest to the surface of cells. In studies in mice,rat, and guinea pigs, a variety of protein antigens are chemicallyattached to the surface of splenocytes and leukocytes. See, e.g., Milleret al., J Exp Med 1979; Braley-Mullen et al., Cell Immunol 1980; Luo etal., PNAS 2008; Smarr et al., J Immunol 2011.

In a recent phase I clinical study in humans, a cocktail of peptideantigens associated with multiple sclerosis were chemically coupled toautologous peripheral blood mononuclear cells and reinfused intopatients (Lutterotti and Martin, Sci Trans Med 2013). The cells werewell tolerated, and there was evidence of a decrease in antigen-specificT cell responses.

EHCs are a prominent source of dying cells. A large number oferythrocytes are cleared after apoptosis-like programmed cell death,called eryptosis, each day (more than 1% per day in humans,approximately 1×10¹¹ cells). Although the exact triggers of erythrocyteclearance remain unclear, eryptotic erythrocytes are characterized byphosphatidylserine asymmetry, membrane heterogeneity, and annexin-Vbinding, analogous to apoptotic nucleated cells.

EHCs are also persistent in the body. Erythrocytes circulate for up to120 days in the adult human. As such, EHCs that comprise an antigen ofinterest may enable the persistent exposure of the antigen to the host.As described above, though the exact molecular mechanisms are not fullyunderstood, it is thought that persistent exposure to antigen can induceperipheral tolerance through antigen presentation in the absence ofcostimulatory signals, leading to the expansion of regulatory T cells,the deletion and anergy of effector T and B cells, and the secretion ofanti-inflammatory and pro-tolerogenic cytokines.

The induction of peripheral tolerance by taking advantage oferythrocytes have been explored experimentally as well. In preliminarywork, the model antigen ovalbumin has been shown to induceantigen-specific CD8 T cell deletion and antigen-specific Treg inductionwhen non-covalently attached to erythrocytes (Kontos et al., PNAS 2013)or osmotically loaded into erythrocytes (Cremel and Godfrin, Int J Pharm2013).

Cultured EHCs of the invention comprising an exogenous antigen ofinterest may have distinct advantages over antigen that isnon-covalently attached to erythrocytes, e.g. via a polypeptide bindingdomain. One advantage may be that the bio-distribution of an EHCcomprising an exogenous antigen of interest is more defined than that ofa polypeptide composition of antigen with targeting domain. The EHCcomprising an exogenous antigen of interest will be confined to thevasculature and to places that erythrocytes typically reside, e.g.spleen. EHCs comprising an exogenous antigen of interest will not befiltered out of the kidney or exit into peripheral tissue, problems thatmay arise when polypeptide antigen compositions are administered. Thedose of exogenous antigen per EHC may be significantly higher if thecells comprising an exogenous antigen of interest are cultured than if apolypeptide antigen is directly injected into the bloodstream anddistributed across approximately 10 trillion erythrocytes in thebloodstream. In some instances, it may be preferable to have theexogenous antigen of interest confined in the intracellular compartmentof the EHC. For example, if the antigen is immunogenic, intracellularlocalization may be advantageous because it may mask the immunogenicantigen from the immune system and thus prevent or reduce immuneactivation. This configuration is not possible with a polypeptideantigen composition.

Cultured EHCs that express an exogenous antigen of interest may havedistinct advantages over antigen that is osmotically loaded into EHCs.The cultured EHCs comprising an exogenous antigen of interest will havecell membranes and cytoskeleton that are substantially unaltered, incontrast to the product of an osmotic loading procedure, in which largepores breach the integrity of the cell membrane and cytoskeleton. Themorphology and biophysical characteristics of EHCs are crucialdeterminants of the cell's bio-distribution, circulation, andinteraction with the vasculature and immune cells (e.g. Pries et al.,Cardiovasc. Res. 1996), and hence maintaining cell integrity may becrucial to retain efficaciousness. Exogenous antigen that is physicallyattached to a cultured EHC, for example by direct fusion to anendogenous cytoplasmic protein or fusion to an endogenous transmembraneprotein, will not leak out of the cell and be exposed to the immunesystem until the EHC is consumed. The problem of leakage may arise ifthe cell is contacted with the antigen using an osmotic loadingprocedure in which the cell membrane can be damaged.

Cultured EHCs that express an antigen of interest can be administereddirectly to a subject in need of the antigen. A separation andpurification of the antigen during manufacture of the product is notrequired. This is in contrast to an osmotic loading product, in whichthe antigen must be synthesized and purified separately and thencombined with the cell, and may provide a significant cost and timeadvantage in the manufacture of the product. The cultured EHCs thatexpress an antigen of interest can be scaled up by propagation inculture. Large, industrial-size batches of cells may be produced togenerate a substantially uniform pharmaceutical composition of EHCs fora given antigen that can be used to universally treat many subjects. Incontrast, osmotic loading is generally limited to aone-donor-to-one-subject scale.

Acquisition of Cells

Exogenous antigen-expressing enucleated hematopoietic cells of theinvention can be generated by any method described herein. In someembodiments, the steps comprise contacting isolated optionally culturedcells derived from hematopoietic stem cells with an antigen.Hematopoietic stem cells give rise to all of the blood cell types foundin mammalian blood including myeloid (monocytes and macrophages,neutorphils, basophils, eosinophils, erythrocytes,megakaryocytes/platelets, dendritic cells) and lymphoid lineages(T-cells, B-cells, NK-cells). Hematopoietic stem cells may be isolatedfrom the bone marrow of adult bones including, for example, femur, hip,rib, or sternum bones. Cells may be obtained directly from the hip, forexample, by removal of cells from the bone marrow using aspiration witha needle and syringe. Alternatively, hematopoietic stem cells may beisolated from normal peripheral blood following pre-treatment withcytokines such as, for example, granulocyte colony stimulating factor(G-CSF). G-CSF mobilizes the release of cells from the bone marrowcompartment into the peripheral circulation. Other sources ofhematopoietic stem cells include umbilical cord blood and placenta.

Isolated hematopoietic stem cells may be cultured, expanded anddifferentiated ex vivo to provide a variety of source material togenerate exogenous antigen-expressing EHCs. For example, hematopoieticstem cells isolated from bone marrow, cytokine-stimulated peripheralblood or umbilical cord blood may be expanded and differentiated ex vivointo mature erythrocytes (Giarratana et al., Nature Biotech. 23:69-74(2005); U.S. Patent Application 2007/0218552). As such, CD34+ cells areisolated from bone marrow or peripheral or cord blood using, forexample, magnetic microbead selection and Mini-MACS columns (MiltenyiBiotech). In one example, the cells are subsequently cultured inmodified serum-free medium supplemented with 1% bovine serum albumin(BSA), 120 μg/ml iron-saturated human transferrin, 900 ng/ml ferroussulfate, 90 ng/ml ferric nitrate and 10 μg/ml insulin and maintained at37° C. in 5% carbon dioxide in air. Expansion and differentiation of thecell culture may occur in multiple steps. For example, in the initialgrowth step following isolation, the cells may be expanded in the mediumdescribed herein in the presence of multiple growth factors including,for example, hydrocortisone, stem cell factor, IL-3, and erythropoietin.In the second stage, the cells may optionally be co-cultured, forexample, on an adherent stromal layer in the presence of erythropoietin.In a third stage, the cells may be cultured on an adherent stromal layerin culture medium in the absence of exogenous factors. The adherentstromal layer may be murine MS-5 stromal cells, for example.Alternatively, the adherent stromal layer may be mesenchymal stromalcells derived from adult bone marrow. The adherent stromal cells may bemaintained in RPMI supplemented with 10% fetal calf serum, for example.In some embodiments, the erythroid precursor cells and cell populationsderived therefrom are not co-cultured with non-EHCs, e.g.,with anadherent stromal layer, i.e. they are cultured in the absence ofnon-EHCs. In some embodiments, EHCs comprising an antigen are culturedin the absence of non-EHCs and are differentiated so that greater than10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or greater than 98% of EHCs are enucleated and thepopulation of enucleated cells is obtained without an enrichment step,such as gravitational separation, magnetic or fluorescent sorting,irradiation, poisoning of nucleated cells, and the like to select forenucleated cells.

In some instances, it may be desirable to expand and partiallydifferentiate the CD34+ hematopoietic stem cells in vitro and to allowterminal differentiation into mature erythrocytes to occur in vivo (See,e.g., Neildez-Nguyen et al., Nature Biotech. 20:467-472 (2002)).Isolated CD34+ hematopoietic stem cells may be expanded in vitro in theabsence of the adherent stromal cell layer in medium containing variousfactors including, for example, Flt3 ligand, stem cell factor,thrombopoietin, erythropoietin, and insulin growth factor. The resultingerythroid precursor cells may be characterized by the surface expressionof CD36 and GPA, and may be transfused into a subject where terminaldifferentiation to mature erythrocytes is allowed to occur.

In some embodiments, the EHC population comprises a plurality ofenucleated EHCs that comprise an antigen polypeptide that is retainedduring enucleation. The resulting isolated enucleated EHC comprising anantigen polypeptide exhibits substantially the same osmotic membranefragility as a corresponding isolated, unmodified, uncultured EHC.

In some embodiments, the EHC population comprises a plurality oferythrocyte precursor cells in substantially the same stage ofdifferentiation and/or cell cycle stage, wherein the precursor cellscomprise a recombinant nucleic acid encoding an antigen. The majority oferythrocyte precursor cells that comprise a recombinant nucleic acidencoding an antigen are capable of differentiating into matureerythrocytes that retain the antigen without retaining the recombinantnucleic acid.

In some embodiments, the primary cells may be collected throughvenipuncture, capillary puncture, or arterial puncture. From thecollected whole blood erythrocytes, platelets or other cells may then beisolated using one, or a combination of techniques including plasmadepletion, density gradient, Hetastarch, PrepaCyte-CB, andcentrifugation.

Allogeneic/and Autologous Sourcing

In some embodiments, generating an exogenousan exogenousantigen-expressing EHC comprises contacting isolated optionally culturedcells that are autologous and/or allogeneic to the subject with anantigen. For example, erythrocytes allogeneic to the subject include oneor more of blood type specific erythrocytes or one or more universaldonor erythrocytes. In some embodiments, exogenous antigen-expressingEHCs may be generated through fusion of erythrocytes, e.g., betweenerythrocytes autologous to the subject and one or more allogeneicerythrocytes, liposomes, and/or artificial vesicles.

In certain embodiments, autologous transfusion of exogenousantigen-expressing EHCs includes isolating erythrocytes, reticulocytesor hematopoietic stem cells from a subject, generating a suitableexogenous antigen-expressing EHC by contacting the cell with an antigenby methods described herein and administering (e.g., by transfusion) theexogenous antigen-expressing EHC into the same subject.

In certain embodiments, allogeneic transfusion of exogenousantigen-expressing EHCs includes isolating erythrocytes, reticulocytesor hematopoietic stem cells from a donor, generating a suitableexogenous antigen-expressing EHC by contacting the cell with an antigenby methods described herein and administering (e.g., by transfusion) theexogenous antigen-expressing EHC into a subject that is different fromthe donor. Where allogeneic cells are used for transfusion, care needsto be taken to use a compatible ABO blood group to prevent an acuteintravascular hemolytic transfusion reaction which is characterized bycomplement activation and lysis of incompatible erythrocytes. The ABOblood types are defined based on the presence or absence of the bloodtype antigens A and B, monosaccharide carbohydrate structures that arefound at the termini of oligosaccharide chains associated withglycoproteins and glycolipids on the surface of the erythrocytes(reviewed in Liu et al., Nat. Biotech. 25:454-464 (2007)). Group Oerythrocytes lack either of these antigenic monosaccharide structures.Subjects with group A erythrocytes have naturally occurring antibodiesto group B erythrocytes whereas subjects with group B erythrocytes haveantibodies to group A erythrocytes. Blood group AB subjects have neitherantibody and blood group O individuals have both. Subjects with eitheranti-A and/or anti-B antibodies cannot receive a transfusion of bloodcontaining the corresponding antigen. Because group O erythrocytescontain neither A nor B antigens, they can be safely transfused intorecipients of any ABO blood group, e.g., group A, B, AB, or Orecipients. Group O erythrocytes are considered universal and may beused in all blood transfusions. In contrast, group A erythrocytes may begiven to group A and AB recipients, group B erythrocytes may be given togroup B and AB recipients, and group AB erythrocytes may only be givento AB recipients. In embodiments in which exogenous antigen-expressingEHCs are generated by contecting erythrocytes or their precursors withan antigen the sourced erythrocytes or their precursors are matched forcompatibility with the recipient.

In some instances, it may be beneficial to convert an exogenousantigen-expressing EHC comprising a non-group O erythrocyte to auniversal blood type. Enzymatic removal of the immunodominantmonosaccharides on the surface of group A and group B erythrocytes maybe used to generate a population of group O-like exogenousantigen-expressing EHCs (See, e.g., Liu et al., Nat. Biotech. 25:454-464(2007)). Group B exogenous antigen-expressing EHCs may be convertedusing an α-galactosidase derived from green coffee beans. Alternativelyor in addition, α-N-acetylgalactosaminidase and α-galactosidaseenzymatic activities derived from E. meningosepticum bacteria may beused to respectively remove the immunodominant A and B antigens (Liu etal., Nat. Biotech. 25:454-464 (2007)), if present on the exogenousantigen-expressing EHCs. In one example, packed red blood cells isolatedas described herein, are incubated in 200 mM glycine (pH 6.8) and 3 mMNaCl in the presence of either α-N-acetylgalactosaminidase andα-galactosidase (about 300 μg/ml packed red blood cells) for 60 min at26° C. After treatment, the red blood cells are washed by 3-4 rinses insaline with centrifugation and ABO-typed according to standard bloodbanking techniques.

In specific embodiments, the exogenous antigen-expressing EHCs describedherein may be generated in the following way. First, erythroid precursorcells are isolated. These cells may alternatively be autologous to thepatient or from substantially universal donor blood. For example, thecells may be ABO type O, rhesus factor Rh r/r, Duffy −/−, and large Kellantigen K1 negative. In the course of differentiation from erythroidprecursor cell to EHC, a recombinant nucleic acid encoding the antigenis introduced. The recombinant nucleic acid encoding the antigen can beunder the control of an erythroid-specific promoter, such as a GATA-1promoter (see e.g., Repik et al., Clin Exp Immunol 2005, 140:230). Therecombinant nucleic acid encoding the antigen can be introduced in anyway known in the art, for example, as plasmid DNA, virus, or mRNA.Nucleic acid introduction can be achieved by a variety of standardmethods, e.g., transfection, transduction, or electroporation.

Platelet Derivation and Maturation

In specific embodiments, the exogenous antigen-expressing EHCs describedherein may be generated by contacting platelets with an antigen. Eachday an adult human produces 2×10¹¹ red blood cells, and about one-halfas many white cells and platelets. In humans, nearly all blood cellproduction occurs in the red bone marrowthat represents a hierarchicaldevelopmental system composed of hematopoietic stem cells, intermediatelevel progenitors and maturing cells committed to each lineage.

Although the morphology of all the major blood cell types is similarthrough their initial development stages, megakaryocytes, cellscommitted to platelet production, are marked by an obvious structuraland functional departure beyond the blast cell level of differentiationgrowing to a size 10 times the diameter of most other bone marrow andblood cells, and containing up to 128 times the normal chromosomalcomplement, these cells give rise to blood platelets. After a series ofnormal cell divisions, the developing megakaryocyte precursor enters aunique cell cycle characterized by a brief (about 1 h) G1 phase, atypical (7 h) S phase, a very brief (˜45 min) G2 phase, followed by theendomitotic phase (an aborted M phase). Once the cell develops a highlypolyploid nucleus, it also develops demarcation membranes necessary forcytoplasmic fragmentation. This event is accompanied by expression ofglycoprotein GPIIbIIIa (platelet fibrinogen receptor; Papayannopoulou etal., Exp. Hematol., 24: 660-9, 1996) and GPIb (von Willibrand factorreceptor; Kaushansky et al., Nature, 369: 568-571, 1994), the granulesthat contain ADP, serotonin, -thromboglobulin, and other substancescritical for mature platelet function. Finally, highly polyploidmegakaryocytes undergo cytoplasmic partitioning, allowing the release ofthousands of platelets (Choi et al., Blood, 85: 402-413, 1995; Cramer etal., Blood, 89: 2336-2346, 1997).

Like all blood cell precursors, megakaryocytes are derived frompluripotent marrow stem cellsthat retain the capacity to extensivelyself-renew, or to differentiate into all of the elements of the blood.Platelet production is in part regulated by signaling mechanisms inducedby interaction between thrombopoietin (TPO) and its cellular receptorTPOR/MPUc-MPL.

Thrombopoietin (TPO) is a hematopoietic growth factor involved instimulation of megakaryocytopoiesis and platelet production. TPO isexpressed in liver and kidney, and, in response to platelet demand, itsexpression may be also upregulated in the bone marrow microenvironment(Kato et al., Stem Cells, 16: 322-328, 1998; McCarty et al., Blood,86:3668-3675, 1995). As TPO expression is mostly constitutive, the TPOlevels are believed to be regulated by sequestering by platelets(Fielder et al., Blood 87: 2154, 1996).

The gene encoding TPO has been cloned and characterized (Kuter et al.,Proc. Natl. Acad. Sci. USA, 91:11104-11108, 1994; Bartley et al., Cell,77:1117-1124, 1994; Kaushansky et al., Nature, 369:568-571, 1994;Wendling et al., Nature, 369:571-574, 1994, and de Sauvage et al.,Nature, 369:533-538, 1994). Human TPO (hTPO) cDNA encodes a 353 aminoacid-long polypeptide. The full-length hTPO secreted from mammaliancells after cleavage of the signal peptide consists of 332 amino acids.Although the predicted molecular mass of this protein is 38 kDa, themolecular masses reported from measurements of material in serum or inculture fluid from recombinant cells vary from 18 to 85 kD(glycosylation, and post-translational proteolytic processing).

The cell surface receptor for TPO (TPOR/MPL/c-MPL) is a product of theprotooncogene c-mpl, a homologue of v-mpl, an envelope protein of themyeloproliferative leukaemia virus (MPLV) shown to induce a pan-myeloiddisorder (Wendling, Virol., 149:242-246, 1986). The human c-mpl genecodes for a protein of 635 aa having a predicted molecular weight of 71kD (Vigon et al., Proc. Natl. Acad. Sci. USA, 89:5640-44, 1992; Mignotteet al., Genomics, 20: 5-12, 1994).

Mice rendered null for the expression of either TPO or its receptor(TPOR/MPL/c-MPL) manifest a severe thrombocytopenic phenotype (Gurney etal., Science, 265: 1445, 1994; Kaushansky et al., J. Clin. Invest., 96:1683, 1995; de Sauvage et al., J. Exp. Med., 183: 651, 1996).

Multiple cytokines (e.g., stem cell factor [SCF], IL-1, IL-3, IL-6,IL-11, leukaemia inhibiting factor [LIF], G-CSF, GM-CSF, M-CSF,erythropoietin (EPO), kit ligand, and -interferon) have been shown topossess thrombocytopoietic activity.

Platelet Activation

The resulting platelets are small disc-shaped cell fragments whichundergo a rapid transformation when they encounter sites of vasculardamage. They become more spherical and extrude pseudopodia, theirfibrinogen receptors are activated leading to aggregation, and theyrelease their granule contents and eventually they form a plug which isresponsible for primary hemostasis (Siess, W., Physiol. Rev. 69: 58-178,1989). Activation of platelets is also implicated in the pathogenesis ofunstable angina, myocardial infarction and stroke (Packham, M. A., CanJ. Physiol Pharmacol. 72: 278-284).

Several physiological substances are involved in the activation ofplatelets such as collagen, which is exposed at the subendothelialsurfaces, thrombin, generated by the coagulation cascade, andthromboxane A2 (TXA₂) and ADP, which are released from activatedplatelets. Collagen binds to several platelet membrane proteinsincluding integrin α2 β1 leading to platelet activation through therelease of TXA₂ and ADP (Shattil, S. J., et al., Curr. Opin. Cell Biol.6: 695-704, 1994). In contrast, thrombin, TXA₂, and ADP, activateG-protein coupled receptors directly and induce platelet aggregation andgranule release (Hourani, S. M, and Cusack, N. J., Pharmacol. Rev. 43:243-298, 1991). The major events involved in platelet activation arebelieved to be the result of the activation of β-isoforms ofphospholipase C (PLC) leading to the generation of inositol 1,4,5triphosphate and diacylglycerol. Platelets mainly contain two isoforms,PLC-β2 and PLC-β3.

Platelet receptors which mediate platelet adhesion and aggregation arelocated on the two major platelet surface glycoprotein complexes. Thesecomplexes are the glycoprotein Ib-IX complex which facilitates plateletadhesion by binding von Willebrand factor (vWF), and the glycoproteinIIb-IIIa complex which links platelets into aggregates by binding tofibrinogen. Patients with the Bernard-Soulier syndrome, a congenitalbleeding disorder, show deficient platelet adhesion due to a deficiencyin the glycoprotein Ib-IX complex which binds vWF, mildthrombocytopenia, and large lymphocoid platelets.

Glycoprotein V (GPV) is a major (≈12,000 molecules/platelet), heavilyglycosylated platelet membrane protein (Mr 82,000). Exposure ofplatelets to thrombin liberates a 69 kDa soluble fragment termed GPVfl.GPV can interact non-covalently with the GPIb-IX complex a complexformed by the non-covalent association of GPIb (consisting of GPIba, a145 kDa protein, disulfide linked to GPIN3, a 24 kDa protein) with GPIX(a 22 kDa protein). The binding sites for von Willebrand factor and forthrombin on the GPIb-IX complex have been localized on GPIba. Sincethrombin is now known to activate platelets by cleaving the thrombinreceptor (Vu et. al., Cell 64:1057-1068 (1990)), a G-protein coupledreceptor, it is unknown whether thrombin cleaves GPV incidently as aconsequence of thrombin binding to GPIbα, or whether this cleavage has aphysiological role. GPIBα, GPIBβ, and GPIX contain one or morehomologous 24 amino acid leucine-rich domains. These domains are alsofound in a large family of leucine-rich glycoproteins (LRG).

GPV is a marker for the megakaryocytic cell lineage. A monoclonalantibody specific for GPV (SW16) does not bind to red cells, leukocyteseendothelial cells, or cell lines such as HEL or MEG-01 which are knownto express platelet megakaryocyte markers.

Mature GPV is composed of 543 amino acids which contain a singletransmembrane domain, a short cytoplasmic domain (16 residues) and alarge extracellular domain with 8 potential N-glycosylation sites.Analysis of the extracellular domain revealed the presence of 15 tandemLeu-rich repeats of 24 amino acids with homology to GPIbα, andidentified a cleavage site for thrombin near the C-terminus withhomology to the Act chain of fibrinogen.

Culturing Conditions

Sources for generating exogenous antigen-expressing EHCs describedherein include circulating cells such as EHCs. A suitable cell sourcemay be isolated from a subject as described herein from patient-derivedhematopoietic or erythroid progenitor cells, derived from immortalizedEHC lines, or derived from induced pluripotent stem cells, optionallycultured and differentiated. Methods for generating erythrocytes usingcell culture techniques are well known in the art, e.g., Giarratana etal., Blood 2011, 118:5071, Huang et al., Mol Ther 2013, epub ahead ofprint September 3, or Kurita et al., PLOS One 2013, 8:e59890. Protocolsvary according to growth factors, starting cell lines, culture period,and morphological traits by which the resulting cells are characterized.Culture systems have also been established for blood production that maysubstitute for donor transfusions (Fibach et al. 1989 Blood 73:100).Recently, CD34+ cells were differentiated to the reticulocyte stage,followed by successful transfusion into a human subject (Giarratana etal., Blood 2011, 118:5071).

Provided herein are culturing methods for EHCs and exogenousantigen-expressing EHCs derived from EHCs. EHCs can be cultured fromhematopoietic progenitor cells, including, for example, CD34+hematopoietic progenitor cells (Giarratana et al., Blood 2011,118:5071), induced pluripotent stem cells (Kurita et al., PLOS One 2013,8:e59890), and embryonic stem cells (Hirose et al. 2013 Stem CellReports 1:499). Cocktails of growth and differentiation factors that aresuitable to expand and differentiate progenitor cells are known in theart. Examples of suitable expansion and differentiation factors include,but are not limited to, stem cell factor (SCF), an interleukin (IL) suchas IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12,CSF, G-CSF, thrombopoietin (TPO), GM-CSF, erythropoietin (EPO), Flt3,Flt2, PIXY 321, and leukemia inhibitory factor (LIF).

EHCs can be cultured from hematopoietic progenitors, such as CD34+cells, by contacting the progenitor cells with defined factors in amulti-step culture process. For example, EHCs can be cultured fromhematopoietic progenitors in a three-step process.

The first step may comprise contacting the cells in culture with stemcell factor (SCF) at 1-1000 ng/mL, erythropoietin (EPO) at 1-100 U/mL,and interleukin-3 (IL-3) at 0.1-100 ng/mL. The first step optionallycomprises contacting the cells in culture with a ligand that binds andactivates a nuclear hormone receptor, such as e.g., the glucocorticoidreceptor, the estrogen receptor, the progesterone receptor, the androgenreceptor, or the pregnane x receptor. The ligands for these receptorsinclude, for example, a corticosteroid, such as, e.g., dexamethasone at10 nM-100 μM or hydrocortisone at 10 nM-100 μM; an estrogen, such as,e.g., beta-estradiol at 10 nM-100 μM; a progestogen, such as, e.g.,progesterone at 10 nM-100 μM, hydroxyprogesterone at 10 nM-100 μM,5a-dihydroprogesterone at 10 nM-100 μM, 11-deoxycorticosterone at 10nM-100 μM, or a synthetic progestin, such as, e.g., chlormadinoneacetate at 10 nM-100 μM; an androgen, such as, e.g., testosterone at 10nM-100 μM, dihydrotestosterone at 10 nM-100 μM or androstenedione at 10nM-100 μM; or a pregnane x receptor ligand, such as, e.g., rifampicin at10 nM-100 μM, hyperforin at 10 nM-100 μM, St. John's Wort (hypericin) at10 nM-100 μM, or vitamin E-like molecules, such as, e.g., tocopherol at10 nM-100 μM. The first step may also optionally comprise contacting thecells in culture with an insulin-like molecule, such as, e.g., insulinat 1-50 μg/mL, insulin-like growth factor 1 (IGF-1) at 1-50 μg/mL,insulin-like growth factor 2 (IGF-2) at 1-50 μg/mL, or mechano-growthfactor at 1-50 μg/mL. The first step further may optionally comprisecontacting the cells in culture with transferrin at 0.1-5 mg/mL.

The first step may optionally comprise contacting the cells in culturewith one or more interleukins (IL) or growth factors such as, e.g.,IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12,granulocyte colony-stimulating factor (G-CSF), macrophagecolony-stimulating factor (M-CSF), granulocyte-macrophagecolony-stimulating factor (GM-CSF), thrombopoietin, fibroblast growthfactor (FGF), platelet-derived growth factor (PDGF), transforming growthfactor beta (TGF-B), tumor necrosis factor alpha (TNF-A), megakaryocytegrowth and development factor (MGDF), leukemia inhibitory factor (LIF),and Flt3 ligand. Each interleukin or growth factor may typically besupplied at a concentration of 0.1-100 ng/mL. The first step may alsooptionally comprise contacting the cells in culture with serum proteinsor non-protein molecules such as, e.g., fetal bovine serum (1-20%),human plasma (1-20%), plasmanate (1-20%), human serum (1-20%), albumin(0.1-100 mg/mL), or heparin (0.1-10 U/mL).

The second step may comprise contacting the cells in culture with stemcell factor (SCF) at 1-1000 ng/mL and erythropoietin (EPO) at 1-100U/mL. The second step may also optionally comprise contacting the cellsin culture with an insulin-like molecule, such as e.g., insulin at 1-50μg/mL, insulin-like growth factor 1 (IGF-1) at 1-50 μg/mL, insulin-likegrowth factor 2 (IGF-2) at 1-50 μg/mL, or mechano-growth factor at 1-50μg/mL. The second step may further optionally comprise contacting thecells in culture with transferrin at 0.1-5 mg/mL. The second may alsooptionally comprise contacting the cells in culture with serum proteinsor non-protein molecules such as, e.g., fetal bovine serum (1-20%),human plasma (1-20%), plasmanate (1-20%), human serum (1-20%), albumin(0.1-100 mg/mL), or heparin (0.1-10 U/mL).

The third step may comprise contacting the cells in culture witherythropoietin (EPO) at 1-100 U/mL. The third step may optionallycomprise contacting the cells in culture with stem cell factor (SCF) at1-1000 ng/mL. The third step may further optionally comprise contactingthe cells in culture with an insulin-like molecule, such as e.g.,insulin at 1-50 μg/mL, insulin-like growth factor 1 (IGF-1) at 1-50μg/mL, insulin-like growth factor 2 (IGF-2) at 1-50 μg/mL, ormechano-growth factor at 1-50 μg/mL. The third step may also optionallycomprise contacting the cells in culture with transferrin at 0.1-5mg/mL. The third step may also optionally comprise contacting the cellsin culture with serum proteins or non-protein molecules such as, e.g.,fetal bovine serum (1-20%), human plasma (1-20%), plasmanate (1-20%),human serum (1-20%), albumin (0.1-100 mg/mL), or heparin (0.1-10 U/mL).

The culture process may optionally comprise contacting cells by a methodknown in the art with a molecule, e.g., a DNA molecule, an RNA molecule,a mRNA, an siRNA, a microRNA, a lncRNA, a shRNA, a hormone, or a smallmolecule, that activates or knocks down one or more genes. Target genescan include, for example, genes that encode a transcription factor, agrowth factor, or a growth factor receptor, including but not limitedto, e.g., GATA1, GATA2, CMyc, hTERT, p53, EPO, SCF, insulin, EPO-R,SCF-R, transferrin-R, insulin-R.

In one embodiment, CD34+ cells are placed in a culture containingvarying amounts of IMDM, FBS, glutamine, BSA, holotransferrin, insulin,dexamethasone, β-estradiol, IL-3, SCF, and erythropoietin, in threeseparate differentiation stages for a total of 22 days.

In one embodiment, CD34+ cells are placed in a culture containingvarying amounts of IMDM, FBS, glutamine, BSA, holotransferrin, insulin,dexamethasone, β-estradiol, IL-3, SCF, and thrombopoietin, in threeseparate differentiation stages for a total of 14 days.

In one embodiment, CD34+ cells are placed in a culture containingvarying amounts of IMDM, FBS, glutamine, BSA, holotransferrin, insulin,dexamethasone, β-estradiol, IL-3, SCF, and GCSF, in three separatedifferentiation stages for a total of 15 days.

In certain embodiments, cells that comprise an exogenous antigen ofinterest may be comprised of or derived from a plurality of circulatingcells including, but not limited to, those listed in Table A. In apreferred embodiment, the circulating cells of the invention are EHCs,such as, e.g. nucleated red blood cells, red blood cell precursors orenucleated red blood cells. For example, the EHCs are a cord blood stemcell, a CD34+ cell, a hematopoietic stem cell (HSC), a spleen colonyforming (CFU-S) cell, a common myeloid progenitor (CMP) cell, ablastocyte colony-forming cell, a burst forming unit-erythroid (BFU-E),a megakaryocyte-erythroid progenitor (MEP) cell, an erythroidcolony-forming unit (CFU-E), a reticulocyte, an erythrocyte, an inducedpluripotent stem cell (iPSC), a mesenchymal stem cell (MSC), apolychtomratic normoblast, an orthochtomratic normoblast, or thoselisted in Table A1, or a combination thereof. In some embodiments, theEHCs are immortal or immortalized cells, for example immortalizederythroblast cells generated by retroviral transduction of CD34+hematopoietic progenitor cells to express Oct4, Sox2, Klf4, cMyc, andsuppress TP53 (e.g. Huang et al., Mol Ther 2013, epub ahead of printSeptember 3).

Erythrocyte compositions are herein provided, wherein a plurality oferythrocytes express an exogenous antigen of interest or a fragmentthereof. The cells may be cultured from patient-derived hematopoietic orerythroid progenitor cells, derived from immortalized EHC lines, orderived from induced pluripotent stem cells. Methods for generatingerythrocytes in cell culture are known in the art, e.g. Giarratana etal., Blood 2011, 118:5071, Huang et al., Mol Ther 2013, or Kurita etal., PLOS One 2013, 8:e59890. Exogenous antigens can be introduced bytransfection of single or multiple copies of genes, transduction with avirus, or electroporation in the presence of DNA or RNA. Methods forexpression of exogenous proteins in mammalian cells are well known inthe art. For example, expression of exogenous factor IX in hematopoieticcells is induced by viral transduction of CD34+ progenitor cells, seeChang et al., Nat Biotechnol 2006, 24:1017.

The erythrocyte compositions described herein may be generated in thefollowing way. First, erythroid precursor cells are isolated. Thesecells may alternatively be autologous to the patient or fromsubstantially universal donor blood. For example, the cells may be ABOtype O, rhesus factor Rh r/r, Duffy −/−, and large Kell antigen K1negative. In the course of differentiation from erythroid precursor cellto EHC, the nucleic acids encoding the exogenous antigen are introduced.The nucleic acid encoding the exogenous antigen can be under the controlof an erythroid-specific promoter, such as a GATA-1 promoter (see e.g.Repik et al., Clin Exp Immunol 2005, 140:230). The nucleic acid encodingthe exogenous antigen, can be introduced in any way known in the art,for example, as plasmid DNA, virus, or mRNA. Nucleic acid introductioncan be achieved by a variety of standard methods, e.g. transfection,transduction, or electroporation.

Modification of Progenitor Cells. Nucleic acids such as DNA expressionvectors or mRNA for producing the antigen of interest may be introducedinto progenitor cells, which can be isolated from an original source orobtained from expanded above via routine recombinant technology asprovided herein. In some instances, the expression vectors can bedesigned such that they can incorporate into the genome of cells byhomologous or non-homologous recombination by methods known in the art.

In some instances, a nucleic acid encoding a polypeptide that canselectively target and cut the genome, for example a CRISPR/Cas9,transcriptional activator-like effector nuclease (TALEN), or zinc fingernuclease, is used to direct the insertion of the nucleic acid payload ofthe expression vector to a particular genomic location, for example theCR1 locus (1q32.2), the hemoglobin locus (11p15.4), or anothererythroid-associated protein including, but not limited to, those listedin Table C.

In some instances, the nucleic acid is an RNA molecule, or a DNAmolecule that encodes for an RNA molecule, that silences or repressesthe expression of a target gene. For example, the molecule can be asmall interfering RNA (siRNA), an antisense RNA molecule, or a shorthairpin RNA (shRNA) molecule.

Methods for transferring expression vectors into progenitor cellsinclude, but are not limited to, viral mediated gene transfer, liposomemediated transfer, transformation, gene guns, transfection andtransduction, e.g., viral mediated gene transfer such as the use ofvectors based on DNA viruses such as adenovirus, adenoassociated virusand herpes virus, as well as retroviral based vectors. Examples of modesof gene transfer include e.g., naked DNA, CaPO4 precipitation, DEAEdextran, electroporation, protoplast fusion, lipofection, and cellmicroinjection.

Any of the genetically modified progenitor cells described herein can becultured under suitable conditions allowing for differentiation intomature enucleated red blood cells, e.g., the in vitro culturing processdescribed herein. The resulting enucleated red blood cells display andexpress proteins associated with mature erythrocytes, e.g. hemoglobin,glycophorin A, which can be validated and quantified by standard methods(e.g., Western blotting or FACS analysis).

Strategies for Exogenous Antigen Expression

Provided herein are antigens that are exhibited by exogenousantigen-expressing EHCs. In some embodiments, an antigen is capable ofinteracting with a target, e.g., to associate with or bind to a target.An antigen can comprise or may consist essentially of a polypeptide. Insome embodiments, the antigen comprises a polypeptide, a carbohydrate, anucleic acid, a lipid, a small molecule, or a combination thereof. Insome embodiments antigens do not interact with a target but act aspayloads to be delivered by the exogenous antigen-expressing EHC to acell, tissue or other site in the body of a subject.

Antigen Polypeptides, Chimeras and Fusions

In some embodiments, antigens comprise polypeptides. Reciverpolypeptides may range in size from 6 amino acids to 3000 amino acidsand may exceed 6, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,150, 200, 300, 400 or may exceed 500 amino acids. Reciver polypeptidesmay range in size from about 20 amino acids to about 500 amino acids,from about 30 amino acids to about 500 amino acids or from about 40amino acids to about 500 amino acids.

In some embodiments, the antigen polypeptide comprises a chimeric orfusion protein which may comprise two or more distinct protein domains.These chimeric antigens are heterologous or exogenous in the sense thatthe various domains are derived from different sources, and as such, arenot found together in nature and can be encoded e.g., by recombinantnucleic acids. Antigen polypeptides can be produced by a number ofmethods, many of which are well known in the art and also describedherein. For example, antigen polypeptides can be obtained by extraction(e.g., from isolated cells), by expression of a recombinant nucleic acidencoding the antigen polypeptide, or by chemical synthesis. Antigenpolypeptides can be produced by, for example, recombinant technology,and expression vectors encoding the polypeptide introduced into hostcells (e.g., by transformation or transfection) for expression of theencoded antigen polypeptide.

There are a variety of conservative changes that can generally be madeto an amino acid sequence without altering activity. These changes aretermed conservative substitutions or mutations; that is, an amino acidbelonging to a grouping of amino acids having a particular size, chargeor other characteristic can be substituted for another amino acid.Substitutions for an amino acid sequence may be selected from othermembers of the class to which the amino acid belongs. For example, thenonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan, methionine, and tyrosine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine and glutamine The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Such alterations are not expected to substantially affectapparent molecular weight as determined by polyacrylamide gelelectrophoresis or isoelectric point. Conservative substitutions alsoinclude substituting optical isomers of the sequences for other opticalisomers, specifically D amino acids for L amino acids for one or moreresidues of a sequence. Moreover, all of the amino acids in a sequencemay undergo a D to L isomer substitution. Exemplary conservativesubstitutions include, but are not limited to, Lys for Arg and viceversa to maintain a positive charge; Glu for Asp and vice versa tomaintain a negative charge; Ser for Thr so that a free ˜OH ismaintained; and Gln for Asn to maintain a free NH2. Moreover, pointmutations, deletions, and insertions of the polypeptide sequences orcorresponding nucleic acid sequences may in some cases be made without aloss of function of the polypeptide or nucleic acid fragment.Substitutions may include, e.g., 1, 2, 3, or more residues. Any teachingof a specific amino acid sequence or a recombinant nucleic acid encodingthe polypeptide or teaching of the name of the name thereof includes anyconservative substitution point mutations, deletions, and insertions ofthose polypeptide sequences or corresponding nucleic acid sequences andany sequence depositied for the protein or gene in a database that canbe made without a loss of function of the polypeptide or nucleic acidfragment.

In some embodiments, the antigen polypeptide is associated with themembrane of the exogenous antigen-expressing EHC. In other embodiments,the antigen polypeptide is not associated with the membrane of theexogenous antigen-expressing EHC.

In one embodiment the mass ratio of lipid to antigen in the exogenousantigen-expressing EHC is less than 1:1000, approximately 1:1000,approximately 1:500, approximately 1:250, approximately 1:100,approximately 1:50, approximately 1:25, approximately 1:10,approximately 1:9, approximately 1:8, approximately 1:7, approximately1:6, approximately 1:5, approximately 1:4, approximately 1:3,approximately 1:2, approximately 1:1, approximately 2:1, approximately3:1, approximately 4:1, approximately 5:1, approximately 6:1,approximately 7:1, approximately 8:1, approximately 9:1, approximately10:1, approximately 25:1, approximately 50:1, approximately 100:1,approximately 250:1, approximately 500:1, approximately 1000:1,approximately 10,000:1, approximately 100,000:1, approximately1,000,000:1, approximately 10,000,000:1, approximately 100,000,000:1,approximately 1,000,000,000:1 or greater than approximately1,000,000,000:1.

In one embodiment the mass ratio of non-exogenous antigen polypeptide toantigen in the exogenous antigen-expressing EHC is less than 1:1000,approximately 1:1000, approximately 1:500, approximately 1:250,approximately 1:100, approximately 1:50, approximately 1:25,approximately 1:10, approximately 1:9, approximately 1:8, approximately1:7, approximately 1:6, approximately 1:5, approximately 1:4,approximately 1:3, approximately 1:2, approximately 1:1, approximately2:1, approximately 3:1, approximately 4:1, approximately 5:1,approximately 6:1, approximately 7:1, approximately 8:1, approximately9:1, approximately 10:1, approximately 25:1, approximately 50:1,approximately 100:1, approximately 250:1, approximately 500:1,approximately 1000:1, approximately 10,000:1, approximately 100,000:1,approximately 1,000,000:1, approximately 10,000,000:1, approximately100,000,000:1, approximately 1,000,000,000:1 or greater thanapproximately 1,000,000,000:1.

In certain embodiments, the polypeptide antigen is located on thesurface and is exposed to the environment around the exogenousantigen-expressing EHC. In some embodiments, the polypeptide antigen islocated inside and faces the unexposed side of the exogenousantigen-expressing EHC.

In certain embodiments, the polypeptide antigen comprises at least oneof the following domains, an S domain (surface), an A domain (anchor),and/or a U domain (unexposed), wherein the S domain is a surface domainexposed to the environment around the exogenous antigen-expressing EHC,wherein the A domain is an anchor, and wherein the U domain is locatedwithin and/or faces the unexposed side of the exogenousantigen-expressing EHC.

Optionally the antigen polypeptide comprises i) one or more additional Sdomains, termed S′ domains, or ii) one or more additional U domains,termed U′ domains.

In some embodiments, the S domain and the A domain form part of the samepolypeptide chain.

In some embodiments, the A domain and the U domain form part of the samepolypeptide chain.

In some embodiments, any one or more of the S, A, U domain is added tothe exogenous antigen-expressing EHC externally.

In some embodiments, any one or more of the S, A, U domain is producedwithin the exogenous antigen-expressing EHC.

In some embodiments, any one or more of the S, A, U domain is apolypeptide.

In some embodiments, any one or more of the S, A, U domain is not apolypeptide.

Schematics of exemplary conformations of antigens within or on exogenousantigen-expressing EHCs are shown in FIGS. 13A, 13B, and 13C.

The A Domain

In certain embodiments, the A domain is a membrane polypeptide. The Adomain can be, e.g., an integral membrane polypeptide or a membraneassociated polypeptide.

The A domain may be selected from one of the following classes,including but not limited to, for example, alpha-helical bitopic,alpha-helical polytopic, beta-barrel transmembrane, all alphamonotopic/peripheral, all beta monotopic/peripheral, alpha/betamonotopic/peripheral, alpha+beta monotopic/peripheral, alpha helicalpeptides, beta-hairpin peptides, beta-helical peptides, type 1transmembrane protein (N-terminus extracellular), type 2 transmembraneprotein (N-terminus intracellular), type 3 transmembrane protein, type4A transmembrane protein, type 4B transmembrane protein, lipid-anchoredprotein, glycosylphosphatidylinositol (GPI) anchored protein, prenylchain anchored protein, or peptides of nonregular structure.

In certain embodiments, the A domain is endogenous, e.g., endogenous toan EHC, a platelet, or a hematopoietic cell. In some embodiments, the Adomain is endogenous to a mammalian cell.

In certain embodiments, the A domain is exogenous, e.g., exogenous to anEHC, a platelet, or a hematopoietic cell. In some embodiments, the Adomain is exogenous to a mammalian cell.

The A domain may be selected from the the following molecules orfragments thereof, including but not limited to, CD1, CD2, CD3, CD4,CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12w, CD13, CD14,CD15, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26,CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38,CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49a,CD49b, CD49c, CD49d, CD49e, CD49f, CD53, CD54, CD55, CD56, CD57, CD58,CD59, CD61, CD62E, CD62L, CD62P, CD63, CD68, CD69, CD71, CD72, CD73,CD74, CD80, CD81, CD82, CD83, CD86, CD87, CD88, CD89, CD90, CD91, CD95,CD96, CD100, CD103, CD105, CD106, CD107, CD107a, CD107b, CD109, CD117,CD120, CD122, CD123, CD127, CD132, CD133, CD134, CD135, CD138, CD141,CD142, CD143, CD144, CD147, CD151, CD152, CD154, CD155, CD156, CD158,CD163, CD165, CD166, CD168, CD184, CDw186, CD195, CD197, CDw199, CD209,CD202a, CD220, CD221, CD235a, CD271, CD279, CD303, CD304, CD309, CD326,Ras-Related protein 1A, semaporin 7A precursor, Calcium andintegrin-binding protein 1, 55 kDa erythrocyte membrane protein,Flotillin-1, Flotillin-2, Erythroid membrane-associated protein,eukaryotic translation initiation factor 2C 2, cytochrome b5 reductase,cell division control protein 42 homolog, KIAA1363 protein, band3,annexin VII, aquaporin, Ecto-ADP-ribosyltransferase 4, Kell, LFA-3,soulute carrier family 2 member 1, LGALS3 protein, Urea transporter, Rhblood CE group antigen poypeptide, Rh-associated glycoprotein, Dematin,ABO blood groups, Aquaporin 3, Aubergers, Band 3, Basigin, C41, CD44,Cis AB, Colton antigen, Complement Component 4, CR1, DAF, Diego, Duffy,Hh/Bombay antigen, ii antigen, Indian blood group, Kell, Kidd, Lewisantigen, Lutheran antigen, MNS antigen system, Cost group, Er group,Dematin, Stomatin, Tropomyosin, Glucose transporter, Adducin, Rabphilin,C1 tetrahydrofolate synthase, Vel group, Lan antigen, At antigen, Jrantigen, AnWj antigen, Sd antigen, Batty, Bilkes, Box, Christiansen,HJK, HOFM, JFV, JONEs, Jensen, Katagiri, Livesay, Milne, Oldeide,Peters, Rasmussen, Reid, REIT, SARA, Rhesus blood D group, Aldolase,Tropomodulin, Arginase, Creatine kinase, B-Cam protein, Rap1A,Bennett-Goodspeed, P antigen system, Rh blood groupXg antigen system, XKprotein, Yt/Cartwright antigen system, CD58, Rh, Scianna, Radin, DARC(Duffy), CR1 Knops-McCoy, DAF Cromer, Gerbich (GYPC), CD47, GlycophorinA, Band 3 (AE3), GYPB Ss, C4A, C4B Chido, Rodgers C4 component ofcomplement, HLA Bg HLA class I, RHAG Rh-associated Ammonium transport,Glycoprotein, Colton (Co) Water channel protein, ACHE Cartwright (Yt)Acetylcholinesterase, Glutathione transferase, Glycophorin C, Aquaporin,Erythroblast associated membrane protein, CD44, Synaptobrevin 2,Ribonuclease, Duodenal cytochrome B, ABO glycosyl transferases, CD59,CD44 Indian (In), AnWj Adhesion receptor, MER2, DOK DombrockADP-ribosyltransferase, SEMA7A JMH Putative adhesion receptor, UMOD SdaTamm-Horsfall protein (uromodulin), Diego (Di), Wright (Wr) Anionchannel protein (band 3, AE1), Kidd (Jk) Urea transporter, FUT3 Lewis(Le) alpha(1,3) fucosyltransferase, OK Oka Neurothelin, putativeadhesion molecule, LW Adhesion receptor, FUT2 Secretor (Se) alpha(1,2)fucosyltransferase, FUT1 Hh alpha(1,2) fucosyltransferase, LU Lutheran(Lu) Adhesion receptor, P1 Glycosyltransferase, XK Kx Putativeneurotransmitter transporter, XG Xg formerly called PBDX, MIC2,Hemoglobin, Ankyrin, Spectrin, KEL Kell (forms K,k,Kp,Js)Metalloproteinase, Torkildsen antigen, coenzyme Q10, Rab 35, Ral Abinding protein, Zona pellucida binding protein, Lyn B protein, KIaa1741protein, DC38, Calcium transporting ATPase, GPIX, GPIba, GPIbb, GPV,GPIb-IX-V, GPVI, GPIa/IIa, GPIIb/IIIa, GPV/IIa.

The S Domain

In some embodiments, the S domain is a protein or a polypeptide. Inother embodiments, the S domain is a nucleic acid. In some embodiments,the S domain is a chemical. In certain embodiment the S domain is asmall molecule.

In some embodiments, the S domain is a polypeptide selected from orderived from one or more of the following classes, including but notlimited to, a flexible linker, an epitope tag, an enzyme, a protease, anuclease, an antigen, an antibody-like molecule, a ligand of anantibody, a growth factor, a cytokine, a chemokine, a growth factorreceptor, a cytokine receptor, a chemokine receptor, an enzymaticrecognition sequence, a transpeptidase recognition sequence, a proteaserecognition sequence, a cleavable domain, an intein, a DNA bindingprotein, and RNA binding protein, a complement regulatory molecule, acomplement cascade molecule, a clotting cascade molecule, a chelator, acomplement regulatory domain, an SCR domain, a CCP domain, animmunoglobulin or immunogloblulin-like domain, an armadillo repeat, aleucine zipper, a dealth effector domain, a cadherein repeat, an EFhand, a phosphotyrosine binding domain, a pleckstrin homology domain, anSCR homology 2 domain, a zinc finger domain, a cyclic peptide, acell-penetrating peptide.

In some embodiments, the S domain is a non-polypeptide molecule, forexample a nucleic acid, a carbohydrate, or a small molecule. In someembodiments, the S domain is a nucleic acid selected from one or more ofthe following classes, including but not limited to, a DNA aptamer, anRNA aptamer, an siRNA, a shRNA, a single-strand RNA probe, a singlestrand DNA probe, an mRNA, a chemically modified oligonucleotide. Insome embodiments, the S domain is a small molecule selected from one ormore of the following classes, including but not limited to, a chelator,DOTA, a radionuclide, an isotope, an imaging agent, a fluorescentmolecule, a chemiluminescent molecule, a gas.

The U Domain

In some embodiments, the U domain is a protein or a polypeptide. Inother embodiments, the U domain is a nucleic acid. In some embodiments,the U domain is a chemical. In certain embodiment the U domain is asmall molecule.

In some embodiments, the U domain is a polypeptide selected from orderived from one or more of the following classes, including but notlimited to, a flexible linker, an epitope tag, an enzyme, a protease, anuclease, an antigen, an antibody-like molecule, a ligand of anantibody, a growth factor, a cytokine, a chemokine, a growth factorreceptor, a cytokine receptor, a chemokine receptor, an enzymaticrecognition sequence, a transpeptidase recognition sequence, a proteaserecognition sequence, a cleavable domain, an intein, a DNA bindingprotein, and RNA binding protein, a complement regulatory molecule, acomplement cascade molecule, a clotting cascade molecule, a chelator, acomplement regulatory domain, an SCR domain, a CCP domain, animmunoglobulin or immunogloblulin-like domain, an armadillo repeat, aleucine zipper, a dealth effector domain, a cadherein repeat, an EFhand, a phosphotyrosine binding domain, a pleckstrin homology domain, anSCR homology 2 domain, a zinc finger domain, a cyclic peptide, acell-penetrating peptide, a kinase domain, aphosphatase domain, acytoskeletal protein, a protein that interacts with the cytoskeletalprotein, a G-protein coupled receptor, a tyrosine kinase, an ITIMdomain, an ITAM domain.

In some embodiments, the U domain is a non-polypeptide molecule, forexample a nucleic acid, a carbohydrate, or a small molecule. In someembodiments, the U domain is a nucleic acid selected from one or more ofthe following classes, including but not limited to, a DNA aptamer, anRNA aptamer, an siRNA, a shRNA, a single-strand RNA probe, a singlestrand DNA probe, an mRNA, a chemically modified oligonucleotide. Insome embodiments, the U domain is a small molecule selected from one ormore of the following classes, including but not limited to, a chelator,DOTA, a radionuclide, an isotope, an imaging agent, a fluorescentmolecule, a chemiluminescent molecule, a gas.

Examples of Antigen Polypeptides

Examples of antigen polypeptides include: the polypeptide antigencomprises glycophorin A with HA epitope tag at the N terminus; thepolypeptide antigen comprises the leader sequence of glycophorin A, HAepitope tag, and the body sequence of glycophorin A; the polypeptideantigen comprises complement receptor 1 (CR1); the polypeptide antigencomprises the leader sequence of CR1, HA epitope tag, the body sequenceof CR1; the polypeptide antigen comprises the leader sequence of CR1, HAepitope tag, six SCR domains of LHR-A and LHR-B of CR1, the membraneproximal two SCR domains of CR1, the transmembrane region of CR1, andthe intracellular region of CR1; the polypeptide antigen comprises theleader sequence of CR1, HA epitope tag, nine SCR domains of LHR-A andLHR-B and LHR-C of CR1, the membrane proximal two SCR domains of CR1,the transmembrane region of CR1, and the intracellular region of CR1;the polypeptide antigen comprises the leader sequence of CR1, LHR-A ofCR1, LHR-B of CR1, LHR-C of CR1, the membrane proximal two SCR domainsof CR1, the transmembrane region of CR1, and the intracellular region ofCR1; the polypeptide antigen comprises leader sequence of CR1, LHR-A ofCR1, LHR-B of CR1, LHR-C of CR1, the membrane proximal two SCR domainsof CR1, the transmembrane region and intracellular region of glycophorinA; the polypeptide antigen comprises the leader sequence of glycophorinA, an antibody scFv against hepatitis B surface antigen (scFv), a(Gly3Ser)2 flexible linker, HA epitope tag, and the body of glycophorinA; the polypeptide antigen comprises Kell, a (Gly3Ser)2 flexible linker,HA epitope tag, and scFv; the polypeptide antigen comprises Kell and HAepitope tag; the polypeptide antigen comprises a 71-amino acidN-terminal fragment of Kell and an HA epitope tag; the polypeptideantigen comprises a 71-amino acid N-terminal fragment of Kell, a(Gly3Ser)2 flexible linker, and an HA epitope tag; the polypeptideantigen comprises a 79-amino acid N-terminal fragment of Kell and an HAepitope tag; the polypeptide antigen comprises a 79-amino acidN-terminal fragment of Kell, a (Gly3Ser)2 flexible linker, and an HAepitope tag; the polypeptide antigen comprises a 71-amino acidN-terminal fragment of Kell, a (Gly3Ser)2 flexible linker, scFv, and anHA epitope tag; the polypeptide antigen comprises a 79-amino acidN-terminal fragment of Kell, a (Gly3Ser)2 flexible linker, scFv, and anHA epitope tag; the polypeptide antigen comprises the leader sequence ofCD55, scFv, an HA epitope tag, and the terminal 37 amino acids of CD55;the polypeptide antigen comprises the leader sequence of CD55, an HAepitope tag, and the body of CD55. In one embodiment, the polypeptideantigen comprises the leader sequence of CD59, scFv, an HA epitope tag,and the body of CD59; the polypeptide antigen comprises the leadersequence of CD59, and HA epitope tag, and the body of CD59; thepolypeptide antigen comprises adenosine deaminase and an HA epitope tag;the polypeptide antigen comprises phenylalanine hydroxylase and an HAepitope tag; the polypeptide antigen comprises adenosine deaminase, a(Gly3Ser)2 flexible linker, phenylalanine hydroxylase, and an HA epitopetag; the polypeptide antigen comprises glycophorin A, adenosinedeaminase at the cytoplasmic C terminus, and an HA epitope tag; thepolypeptide antigen comprises glycophorin A, phenylalanine hydroxylaseat the cytoplasmic C terminus, and an HA epitope tag.

In certain embodiments, the antigen is capable or interacting with amacrophage. The antigen polypeptide may comprise one or more of: thecomplement receptor (Rieu et al., J. Cell Biol. 127:2081-2091 (1994)),the scavenger receptor (Brasseur et al., Photochem. Photobiol.69:345-352 (1999)), the transferrin receptor (Dreier et al., Bioconjug.Chem. 9:482-489 (1998); Hamblin et al., J. Photochem. Photobiol. 26:4556(1994)); the Fc receptor (Rojanasakul et al., Pharm. Res. 11:1731-1733(1994)); and the mannose receptor (Frankel et al., Carbohydr. Res.300:251-258 (1997); Chakrabarty et al., J. Protozool. 37:358-364(1990)).

Other antigens capable or interacting with a macrophages include: lowdensity lipoproteins (Mankertz et al., Biochem. Biophys. Res. Commun.240:112-115 (1997); von Baeyer et al., Int. J. Clin. Pharmacol. Ther.Toxicol. 31:382-386 (1993)), very low density lipoproteins (Tabas etal., J. Cell Biol. 115:1547-1560 (1991)), mannose residues and othercarbohydrate moieties (Pittet et al., Nucl. Med. Biol. 22:355-365(1995)), poly-cationic molecules, such as poly-L-lysine (Hamblin et al.,J. Photochem. Photobiol. 26:45-56 (1994)), liposomes (Bakker-Woudenberget al., J. Drug Target. 2:363-371 (1994); Betageri et al., J. Pharm.Pharmacol. 45:48-53 (1993)) and 2-macroglobulin (Chu et al., J. Immunol.152:1538-1545 (1994)).

Provided herein are compositions containing EHCs comprising an antigenhaving functional activities that are either i) not present in nativeEHCs of the same lineage, or ii) present in native EHCs of the samelineage in reduced levels or reduced activity levels as compared to theEHCs comprising the antigen. Such functional activities includecomplement inhibition, immune complex clearance, artificial antigenpresentation, modulation of the coagulation cascade, oxygen transfer,drug delivery, cytotoxin adsorption, avoidance of phagocytosis, andextension of circulation time.

In some embodiments, EHCs have higher levels of a complement receptorpolypeptide, such as CR1, than native EHCs of the same lineage by virtueof comprising a CR-1 antigen. In an alternative embodiment, the EHCscomprising an antigen have higher levels of a complement receptoragonist polypeptide or complement associated polypeptide than nativeEHCs of the same lineage, including but not limited to, the polypeptideslisted in table 6 and table 8. The complement receptor antigenpolypeptide comprises a human Complement Receptor-1 (CR1) polypeptide,variant, or functional fragment thereof. The CR1 antigen polypeptide maybe derived from one or more than one of the native alleles of CR1, e.g.,the A allele (also termed the F allele or CR1*1 allele), the B allele(also termed the S allele or CR1*2 allele), the C allele (also termedthe F′ allele or CR1*3 allele), or the D allele (also termed the CR1*4allele). The sequences and database accession numbers for these nativeforms are provided in table 3. In some embodiments, the CR1 antigenpolypeptide contains a domain of a CR1 polypeptide. For example, the CR1polypeptide may comprise one or more short consensus repeat (SCR)domains, also termed complement control protein (CCP) modules or Sushidomains, e.g., Genbank accession number AAV65577.1. In one embodiment,the CR1 antigen polypeptide comprises one or more Short ConsensusRepeats (SCRs), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or greater than 44 SCRs. Inanother embodiment, the CR1 antigen polypeptide comprises one or morelong homologous repeat (LHR) units of CR1, e.g., LHR-A, LHR-B, LHR-C, orLHR-D, e.g., 1, 2, 3, 4, 5, 6 or greater than 6 LHR domains. In anotherembodiment, the CR1 antigen polypeptide may comprise one or more thanone extracellular domains of CR1 fused to another cell membrane protein,e.g., glycophorin A, glycophorin B, glycophorin C, glycophorin D, kell,band 3, aquaporin 1, glut 1, kidd antigen protein, rhesus antigen,including, but not limited to the cell surface moieties listed in table1 and table 6.

In some embodiments, an EHC contains a recombinant nucleic acid encodinga complement receptor antigen polypeptide, or alternatively or incombination, a complement receptor agonist antigen polypeptide orcomplement associated antigen polypeptide including but not limited to,the polypeptides, and agonists to the polypeptides, listed in table 8.In some embodiments, the EHCs further contain an exogenousdecay-accelerating factor (CD59, GenBank: CAG46523.1) polypeptide, or anexogenous membrane cofactor (CD46, GenBank: BAA12224.1) polypeptide, ora variant or functional fragment thereof, or a combination thereof.

CR1 activities include binding to C3b-containing immune complexes andshuttling of these immune complexes from circulation to liver and spleenmacrophages of the reticuloendothelial system. Upon encounter with cellsof the reticuloendothelial system, the immune complex is endocytosed bythe phyagocytic cell but the red blood cell is spared to continue itscirculation. The removal of the immune complex sometimes results inproteolytic cleavage of CR1 from the surface of the red blood cell. Tomeasure binding activity, one can perform an in vitro binding assaybetween EHCs and immune complexes. To measure sparing of the EHC, onecan perform an in vitro phagocytosis assay with phagocytic cells andimmune complex-loaded EHCs. To measure in vivo clearance of circulatingimmune complexes to the liver, one can perform a clearance andbiodistribution assay using radiolabeled immune complexes.

Provided are compositions containing EHCs containing an antigencomprising a native polypeptide at a level greater than that of ahematopoietic cell of the same lineage not comprising the antigenpolypeptide. For example, populations of EHCs contain antigens, such ascomplement receptor 1 levels at least about 1.1, e.g., 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10000, or more than 10000 times greater than correspondinghematopoietic cells of the same lineage that lack the CR1 antigenpolypeptide. CR1 levels on reticulocytes and erythrocytes are typicallybetween 50-2000 molecules per cell (Lach-Trifilieff, J Immunol 1999,162:7549). Provided are compositions that contain populations of EHCswith CR1 levels of at least about 2500, 5000, 6000, 7000, 8000, 9000,10000, 15000, 20000, 25000, 30000, 40000, 50000, 100000, 200000, 300000,400000, 500000, 600000, 700000, 800000, 900000, 1000000, or more than1000000 molecules per cell. CR1 levels in wild-type and exogenousantigen-expressing EHCs can be measured and quantified by, for example,flow cytometry with antibodies specific for CR1.

Provided herein, in some embodiments, are EHCs comprising an antigen,populations of EHCs comprising an antigen, and compositions of EHCscomprising an antigen. In some embodiments, the antigen interacts with acirculating pathogen, such as a virus or a bacterium. In someembodiments, the EHC expresses a recombinant gene encoding an antibody,scFv, or nanobody specific for the circulating pathogen. The antibody,scFv, or nanobody may be expressed as a fusion protein. In otherembodiments, the antibody, scFv, or nanobody antigen or another antigenwith affinity to circulating pathogens is loaded into or onto the EHC.The antibody, scFv, or nanobody antigen or the other antigen withaffinity to circulating pathogens may be localized intracellularly orextracellularly. In some embodiments, the antigen is specific for aviral or bacterial antigen, such as a surface, envelope or capsidantigen.

Provided herein, in certain embodiments, are EHCs comprising an antigen,populations of EHCs comprising an antigen, and compositions of EHCscomprising an antigen. In some embodiments, the antigen interacts with atoxin, preferably a foreign toxin, such as derived from a pathogen orotherwise from the environment. In some embodiments, the EHC expresses arecombinant gene encoding an antigen comprising an amino acid sequencederived from lipopolysaccharide-binding protein (LBP),bactericidal/permeability-increasing protein (BPI), amyloid P component,or a cationic protein. Toxin-binding antigens may be expressed as afusion protein. In other embodiments, toxin-binding antigens may beloaded into or onto the EHC. Toxin-binding antigens may be localizedintracellularly or extracellularly. In some embodiments, the toxinbinding antigen is specific for a bacterial toxin such as botulinum oranthrax.

Further, exogenous antigen-expressing EHCs may express an antigencapable of enhancing its ability to sequester a target. Potentialsequestration enhancement antigens include the polypeptide transportersincluding, but not limited to, those in table 1.

In one embodiment, the antigen comprises a polypeptide that comprises anamino acid sequence derived from Duffy Antigen Receptor for Chemokines(DARC). In one embodiment, the EHC expresses a recombinant gene encodingan amino acid sequence derived from Duffy Antigen Receptor forChemokines (DARC). The DARC antigen may be expressed as a full-lengthprotein or a fragment thereof. DARC may be expressed as a fusionprotein. In other embodiments, DARC protein is loaded into or onto theEHC. In some embodiments, the loaded DARC is additionally functionalizedor otherwise modified. The DARC antigen molecule may be localizedintracellularly or extracellularly.

DARC was identified as a potent multi-ligand chemokine receptor. DARCbelongs to the family of rhodopsin-like seven-helix transmembraneproteins. Besides erythrocytes DARC is expressed in post capillaryvenular endothelial cells, which are the primary site of leukocytetransmigration in most tissues. DARC provides a highly specific bindingsite for both CC and CXC chemokines. DARC is thought to possess a higheraffinity for ELR motif CXC chemokines. CXC chemokines are neutrophilchemoattractants and may potentially be pro-angiogenic.

Interaction between DARC and CXCL8 has demonstrated a dissociationconstant (Kd) of 5 nmol/L and receptor binding sites estimated at1000-9000 per erythrocyte (Hadley, Blood, 1997) Unlike otherseven-transmembrane chemokine receptors, DARC lacks the highly conservedG protein coupling motif located in the second cytoplasmic loop (Meny,Immunohematology, 2010). DARC is not G-protein coupled and has no knownalternative signaling mechanism. The biological role of DARC is notfully understood. DARC is thought to be a) multi-specific; b) unable toinitiate intracellular signals, and c) chemokines bound to erythrocytesurface are believed to be inaccessible to their normal targetinflammatory cells (Neote, J Biol Chem, 1993). Erythrocytes may play arole in the regulation of inflammatory processes through the presence ofDARC

Inflammatory signaling molecules, such as cytokines, can trigger localand systemic tissue damage when present in high concentrations. Burstsof cytokines are implicated in the pathogenesis of bacterial sepsis,rheumatoid arthritis, and several other inflammatory diseases. ModifiedEHCs that exogenously express natural cytokine receptors or syntheticantibody-like receptor mimics can sequester the inflammatory cytokines.An exemplary chemokine receptor is DARC. Provided herein are EHCscomprising an antigen that is a cytokine receptor or chemokine receptor,including, but not limited to DARC. For example, EHCs expressing DARCantigen (thereby increasing the amount present on native erythrocytes)may be used to modulate chemokine levels in circulation and/or withinthe body's peripheral tissues. The EHCs comprising a DARC antigen caneither be marked for destruction or can slowly release the inflammatorymediators back into circulation, but at a low and diffuse concentration.The EHC comprising an antigen that comprises a chemokine or cytokinereceptor may act as a reservoir for signal transduction peptides.

In one embodiment, the antigen comprises a polypeptide that comprises anamino acid sequence derived from an antibody. In one embodiment, the EHCexpresses a recombinant gene encoding an amino acid sequence derivedfrom an antibody. The antibody antigen may be expressed as a full-lengthprotein or a fragment thereof. The antibody may be expressed as a fusionprotein. In other embodiments, the antibody protein is loaded into oronto the EHC. In some embodiments, the loaded antibody is additionallyfunctionalized or otherwise modified. The antibody antigen may belocalized intracellularly or extracellularly. In one embodiment, theantigen comprises an antibody amino acid sequence that is specific for adesired target. In some embodiments, the antibody is a scFv. In otherembodiments, the antibody is a nanobody.

In certain embodiments, the EHCs comprise an antigen that comprises anantibody or fragment thereof that is specific for a target and islocated on the cell surface. For example, a variable fragment (Fv) of anantibody specific for botulinum toxin binding is expressed on thesurface of the EHC. Botulinum toxin binding antibodies are known in theart (Amersdorfer, Inf and Immunity, 1997), as is the expression of theFv portion of an antibody (Hoedemaeker, Journ of Bio Chemistry, 1997).Upon binding, the toxin is retained by the EHC through the Fv region,sequestered and shuttled via the circulatory system to the liver forclearance from the body.

In one embodiment, the antigen comprises a polypeptide that comprises anamino acid sequence derived from a scFv antibody. In one embodiment, theEHC expresses a recombinant gene encoding an amino acid sequence derivedfrom a scFv antibody. The scFv antibody antigen may be expressed as afull-length protein or a fragment thereof. The scFv antibody may beexpressed as a fusion protein. In other embodiments, the scFv protein isloaded into or onto the EHC. Suitable scFv antigen polypeptides that maybe expressed by EHCs include, but are not limited to, those listed intable 6.

scFv antibodies have been constructed mainly from hybridoma, spleencells from immunized mice, and B lymphocytes from human. The variableregion of an antibody is formed by the noncovalent heterodimer of thevariable domains of the V(H) and V(L) domains, which can then be used inthe construction of a recombinant scFv antibody.

The production of scFvs is known in the art and require mRNA to first beisolated from hybridoma (or also from the spleen, lymph cells, and bonemorrow) followed by reverse transcription into cDNA to serve as atemplate for antibody gene amplification (PCR). With this method, largelibraries with a diverse set of antibody-derived scFvs (a set comparableto that of the original antibodies from which the scFvs are modeled) canbe created.

The scFv antigen may be made specific to any target molecule including,but not limited to, those in table 4.

In one example, a scFv antigen specific for anthrax toxin may beexpressed on a EHC. Upon administration to a subject in need thereof aneffective dose of a population of EHC comprising an antigen moleculespecific for anthrax toxin can be used to capture and sequester theanthrax toxin. The EHC migrates to the liver where clearance occurs.

In certain embodiments, erythrocytes comprise an antigen comprising acamelid-derived nanobody expressed on the surface of the cell.Nanobodies are usually 12-15 kDa. They are considerably smaller thanantibodies and scFv. Nanobodies may thus be easier to transfect, and thenanobody antigen will be more easily expressed, translated and ortransported to the cell surface in an EHC. In certain embodiments,nanobody antigens are employed to minimize immunogenic effects caused bya specific antigen. Nanobodies because of their small size will offerreduced immunogenic potential. In certain embodiments, antigennanobodies are employed because they limit changes in the mechanical andmorphological behavior of the plasma membrane of the EHC. This may allowthe EHC to exhibit normal circulatory red blood cell behavior. Incertain embodiments, antigen nanobodies are employed because they havean increased ability to recognize hidden or uncommon epitopes comparedto standard antibodies. For example, they can bind to small enzymaticcavities of a target and modulate the molecular behavior of the target.

In certain embodiments, EHCs comprise antigen nanobodies withspecificity to target epitopes of molecules in the human complementsystem. Such EHCs may be administered to a subject in need thereof toselectively deplete one or more over-active factors of the complementsystem. For example, C5 may be targeted by EHCs comprising antigennanobodies with specificity to target epitopes of C5 and cleared fromthe system by the EHCs upon administration of the cells into a subject.This approach is suitable to provide a therapeutic effect, e.g., for acomplement disorder, such as paroxysmal nocturnal hemoglobinuria. Incertain embodiments, EHCs comprise antigen nanobodies with specificityto target epitopes of molecules including, but not limted to, thoselisted in table 4.

In some embodiments, the antigen comprises a polypeptide that comprisesan amino acid sequence derived from one of proteases, nucleases,amylase, lyase (sucrase) or hydrolase (DNase, lipase). In oneembodiment, the EHC expresses a recombinant gene encoding an amino acidsequence derived from one of proteases, nucleases, amylase, lyase(sucrase) or hydrolase (DNase, lipase). Antigen proteases, nucleases,amylases, lyases and hydrolases may be expressed as a full-lengthprotein or a fragment thereof. Antigen proteases, nucleases, amylases,lyases and hydrolases may be expressed as a fusion protein. In otherembodiments, antigen proteases, nucleases, amylases, lyases orhydrolases are loaded into or onto the EHC. In some embodiments, theloaded antigen proteases, nucleases, amylases, lyases or hydrolases areadditionally functionalized or otherwise modified. The antigen protease,nuclease, amylase, lyase or hydrolase antigen molecule may be localizedintracellularly or extracellularly.

In certain embodiments, EHCs comprise an antigen comprising a protease,a nuclease, an amylase, a lyase or a hydrolase. The EHC comprising aprotease, a nuclease, an amylase, a lyase or a hydrolase antigen iscapable of degrading a target on the EHC independent of circulatoryclearance, e.g., by macrophages in the liver. In certain embodiments,EHCs comprising an antigen comprising a protease, a nuclease, anamylase, a lyase or a hydrolase may be administered to a subject in needthereof to treat a cancer by selectively degrading metabolites that areessential for cancer cell growth. For example, asparaginase is used todecrease local asparagine levels to treat acute lymphoblastic leukemiaand acute myeloid leukemia. Suitable antigens may, e.g., comprise one orboth of the two major classes of enzymes capable of degrading targetmolecules, lyases and hydrolases. In certain embodiments, EHCs areprovided comprising an antigen comprising a molecule including but notlimited to those listed in table 6.

In certain embodiments, erythrocytes comprise an antigen comprising alyase. In one embodiment, the lyase is valine decarboxylase. Valinedecarboxylase antigen may be expressed within the intracellular space ofthe EHC. EHCs comprising a valine decarboxylase antigen may beadministered to a subject in need thereof to modulate valine levelswithin the blood. EHCs comprising a valine decarboxylase antigen aresuitable to treat valinemia, an inherited disorder that increases levelsof the amino acid valine in the blood. Affected individuals typicallydevelop vomiting, failure to thrive, intellectual disability, andfatigue. Valinemia is caused by a deficiency of the valine transaminaseenzyme and has an autosomal recessive pattern of inheritance.

In certain embodiments, erythrocytes comprise an antigen comprising ahydrolase. In one embodiment, the hydrolase is deoxyribonuclease I(DNase I). DNase I antigen may be expressed on the surface of the EHC.EHCs comprising a DNase I antigen may be administered to a subject inneed thereof to preferentially cleave circulating DNA at phosphodiesterlinkages adjacent to a pyrimidine nucleotide, yielding5′-phosphate-terminated polynucleotides with a free hydroxyl group onposition 3′. On average tetra-nucleotides are produced. EHCs comprisinga DNase I antigen are suitable to treat conditions exacerbated by highlevels of immunogenic DNA in circulation, such as systemic lupuserythematosus (SLE).

In certain embodiments the antigen is capable of responding to anexternal stimulus, e.g., upon binding to a ligand or contacting thestimulus, wherein responding entails, for example, moving, re-folding,changing conformation, forming a dimer, forming a homodimer, forming aheterodimer, forming a multimer, transducing a signal, emitting energyin a detectable form (e.g., fluorescence), functionally interacting withanother antigen, or functionally interacting with a non-exogenousantigen polypeptide.

Targets

Provided herein are exogenous antigen-expressing EHCs comprising anexogenous antigen polypeptide capable of interacting with a target.Further provided herein are exogenous antigen-expressing EHCs comprisinga non-polypeptide exogenous antigen capable of interacting with atarget. The exogenous antigen-expressing EHCs may be administered to asubject in need thereof to modulate the amount or concentration of atarget residing in the circulatory system of the subject. A suitableexogenous antigen may be chosen to interact with a specific target.Suitable targets include entities that are associated with a specificdisease, disorder, or condition. However, targets may also be chosenindependent of a specific disease, disorder, or condition.

In some embodiments, the target is an antibody or antibody-likemolecule, for example an autoimmune or a self-antibody, or a foreignantibody, or a therapeutic antibody, including but not limited to, e.g.,an antibody against beta-2 glycoprotein 1, an antibody against I/iantigen, an antibody against the NC1 domain of collagen a3(IV), anantibody against platelet glycoprotein, an antibody againstphospholipase A2 receptor, an antibody against erythrocyte glycophorinA, B, or C, or an antibody against erythrocyte Rh antigen.

In some embodiments, the target is a molecule of the complement cascade,for example C1, C1r, C1s, C1q, C2, C2a, C2b, C3, C3a, C3b, C4, C4b, C4a,C3bBb, C3bBb3b, C4b2b, C4b2b3b, C5, C5a, C5b, C6, C7, C8, C9, poly-C9,membrane attack complex. Factor B, Factor D, Properdin, C3, C3a, C3b,iC3b, C3c, C3dg, C3dk, C3e, Bb, Factor I, C1q, C1r, C1s, C4, C4a, C4b,C2, C4bp, Mannose-Binding Lectin (MBL), MBL-Associated Serine Protease 1(MASP1), MBL-Associated Serine Protease 2 (MASP2), C5, C5a, C6, C7, C8,C9, CR1, CR2, CR3, CR4, C3aR, C3eR, Decay-accelerating factor (DAF),Membrane cofactor protein (MCP), CD59, C3 Beta chain Receptor, C1inhibitor, C4 binding protein, Factor I, Factor H.

In some embodiments, the target is an immune complex, for example an IgGimmune complex, an IgA immune complex, an IgM immune complex.

In some embodiments,the target is an amyloid placque, for example aplacque comprised of beta amyloid, IAPP (Amylin), alpha-synuclein,PrPSc, huntingtin, calcitonin, atrial natriuretic factor, apolipoproteinAI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta 2microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin lightchain AL, S-IBM.

In some embodiments, the target is a bacterium, for exampleEnterococcus, Streptococcus, or Mycobacteria, Rickettsia, Mycoplasma,Neisseria meningitides, Neisseria gonorrheoeae, Legionella, Vibriocholerae, Streptococci, Staphylococcus aureus, Staphylococcusepidermidis, Pseudomonas aeruginosa, Corynobacteria diphtheriae,Clostridium spp., enterotoxigenic Eschericia coli, and Bacillusanthracis. Other pathogens for which bacteremia has been reported atsome level include the following: Rickettsia, Bartonella henselae,Bartonella quintana, Coxiella burnetii, chlamydia, Mycobacterium leprae,Salmonella; shigella; Yersinia enterocolitica; Yersiniapseudotuberculosis; Legionella pneumophila; Mycobacterium tuberculosis;Listeria monocytogenes; Mycoplasma spp.; Pseudomonas fluorescens; Vibriocholerae; Haemophilus influenzae; Bacillus anthracis; Treponemapallidum; Leptospira; Borrelia; Corynebacterium diphtheriae;Francisella; Brucella melitensis; Campylobacter jejuni; Enterobacter;Proteus mirabilis; Proteus; and Klebsiella pneumoniae.

In some embodiments, the target is a virus, including but limited to,those whose infection involves injection of genetic materials into hostcells upon binding to cell surface receptors, viruses whose infection ismediated by cell surface receptors. Non-limiting examples of theseviruses can be selected from Paramyxoviridae (e.g., pneumovirus,morbillivirus, metapneumovirus, respirovirus or rubulavirus),Adenoviridae (e.g., adenovirus), Arenaviridae (e.g., arenavirus such aslymphocytic choriomeningitis virus), Arteriviridae (e.g., porcinerespiratory and reproductive syndrome virus or equine arteritis virus),Bunyaviridae (e.g., phlebovirus or hantavirus), Caliciviridae (e.g.,Norwalk virus), Coronaviridae (e.g., coronavirus or torovirus),Filoviridae (e.g., Ebola-like viruses), Flaviviridae (e.g., hepacivirusor flavivirus), Herpesviridae (e.g., simplexvirus, varicellovirus,cytomegalovirus, roseolovirus, or lymphocryptovirus), Orthomyxoviridae(e.g., influenza virus or thogotovirus), Parvoviridae (e.g.,parvovirus), Picomaviridae (e.g., enterovirus or hepatovirus),Poxviridae (e.g., orthopoxvirus, avipoxvirus, or leporipoxvirus),Retroviridae (e.g., lentivirus or spumavirus), Reoviridae (e.g.,rotavirus), Rhabdoviridae (e.g., lyssavirus, novirhabdovirus, orvesiculovirus), and Togaviridae (e.g., alphavirus or rubivirus).Specific examples of these viruses include human respiratorycoronavirus, influenza viruses A-C, hepatitis viruses A to G, and herpessimplex viruses 1-9.

In some embodiments, the target is a parasite, including but not limitedto, for example, intestinal or blood-borne parasites, protozoa,trypanosomes; haemoprotozoa and parasites capable of causing malaria;enteric and systemic cestodes including taeniid cestodes; entericcoccidians; enteric flagellate protozoa; filarial nematodes;gastrointestinal and systemic nematodes and hookworms.

In some embodiments, the target is a fungus, including but not limitedto, for example, Candida albicans, Candida glabrata, Aspergillus, T.glabrata, Candida tropicalis, C. krusei, and C. parapsilosis.

In some embodiments, the target is a bacterial toxin, including but notlimited to, for example, AB toxin, alpha toxin, anthrax toxin,bacteriocin, botunlinum toxin, cholesterol-dependent cytolysin,Clostridium botulinum C3 toxin, Clostridium difficile toxin A,Clostridium difficile toxin B, Clostridium enterotoxin, Clostridiumperfringens alpha toxin, Clostridium perfringens beta toxin, Cordfactor, Cry1Ac, Cryptophycin, Delta endotoxin, Diphtheria toxin,Enterotoxin type B, erythrogenic toxin, exfoliatin, haemolysin E,heat-labile enterotoxin, heat-stable enterotoxin, hemolysin, leukocidin,lipopolysaccharide, Listeriolysin O, microcin, Panton-Valentineleucocidin, pathogenicity island, phenol-soluble modulin, pneumolysin,pore-forming toxin, Pseudomonas exotoxin, RTX toxin, sakacin,Staphylococcus aureus alpha toxin, Staphylococcus aureus beta toxin,Staphylococcus aureus delta toxin, Streptolysin, Symplocamide A,tabtoxin, tetanolysin, tetanospasmin, thiol-activated cytolysin,tolaasin, toxic shock syndrome toxin, toxoflavin, trehalose dimycolate,verocytotoxin, and vibriocin.

In some embodiments, the target is a prion protein, including but notlimited to, for example, PRP, PRPc, PRPsc, PRPres.

In some embodiments, the target is a cytokine or a chemokine or a growthfactor, including but not limited to, for example, acylation stimulatingprotein, adipokine, albinterferon, CCL1, CCL11, CCL12, CCL13, CCL14,CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23,CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL5, CCL6, CCL7, CCL8, CCL9,colony-stimulating factor, CX3CL1, CX3CR1, CXCL1, CXCL10, CXCL11,CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2, CXCL3, CXCLS, CXCL6,CXCL7, CXCL9, erythropoietin, Gc-MAF, granulocyte colony-stimulatingfactor, granulocyte macrophage colony-stimulating factor, hepatocytegrowth factor, IL 10 family, IL 17 family, ILIA, IL1B, interferon,interferon beta 1a, interferon beta 1b, interferon gamma, interferontype I, interferon type II, interferon type III, interleukin,interleukin 1 family, interleukin 1 receptor antagonist, interleukin 10,interleukin 12, interleukin 12 subunit beta, interleukin 13, interleukin16, interleukin 2, interleukin 23, interleukin 23 subunit alpha,interleukin 34, interleukin 35, interleukin 6, interleukin 7,interleukin 8, interleukin-36, leukemia inhibitory factor,leukocyte-promoting factor, lymphokine, lymphotoxin, lymphotoxin alpha,lymphotoxin beta, macrophage colony-stimulating factor, macrophageinflammatory protein, macrophage-activating factor, monokine, myokine,myonectin, nicotinamide phosphoribosyltransferase, oncostatin M,oprelvekin, platelet factor 4, proinflammatory cytokine, promegapoietin,RANKL, stromal cell-derived factor 1, talimogene laherparepvec, tumornecrosis factor alpha, tumor necrosis factors, XCL1, XCL2, XCR1,angiopoietin, basic fibroblast growth factor, betacellulin, bonemorphogenetic protein, brain-derived neurotrophic factor, CCNintercellular signaling protein, CTGF, darbepoetin alfa, endoglin,epidermal growth factor, epoetin alfa, epoetin beta, erythropoietin,FGF15, FGF15/19, fibroblast growth factor, fibroblast growth factor 23,filgrastim, GLIA maturation factor, granulocyte colony-stimulatingfactor, granulocyte macrophage colony-stimulating factor, growthdifferentiation factor-9, heberprot-P, hemopoietic growth factors,heparin-binding EGF-like growth factor, hepatocyte growth factor,insulin-like growth factor, insulin-like growth factor 1, insulin-likegrowth factor 2, keratinocyte growth factor, myostatin, nerve growthfactor, neurotrophin-3, neurotrophin-4, oncomodulin, osteopromotive,palifermin, PDGFB, placental growth factor, platelet alpha-granule,platelet-derived growth factor, platelet-derived growth factor receptor,proliferative index, thrombopoietin, transforming growth factor,vascular endothelial growth factor.

In some embodiments, the target is a small molecule, for example achemical, an amino acid, an atom, an element, an organic acid, <2000 Da,<1000 Da, <500 Da, including but not limited to, for example, iron,copper, calcium, potassium, ethanol, methanol, glycine, alanine, valine,leucine, isoleucine, serine, cysteine, selenocysteine, threonine,methionine, proline, phenylalanine, tyrosine, tryptophan, histidine,lysine, arginine, aspartate, glutamate, asparagine, glutamine

In some embodiments, the target is a lipid, lipid complex, proteolipidcomplex, or cholesterol, including but not limited to for example, LDL,VLDL, HDL, HDL2B, triglycerides, LP(a), cholesterol.

In some embodiments, the target is a mammalian cell, including but notlimited to, for example, a human cell, a circulating cell, an immunecell, a neutrophil, an eosinophil, a basophil, a lymphocyte, a monocyte,a B cell, a T cell, a CD4+ T cell, a CD8+ T cell, a gamma-delta T cell,a regulatory T cell, a natural killer cell, a natural killer T cell, amacrophage, a Kupffer cell, a dendritic cell, a cancer cell, a cancerstem cell, a circulating tumor cell, a cancer cell from one of thefollowing cancers including, but not limited to,

ACUTE lymphoblastic leukaemia (ALL), ACUTE myeloid leukaemia (AML), analcancer, bile duct cancer, bladder cancer, bone cancer, bowel cancer,brain tumours, breast cancer, cancer of unknown primary, cancer spreadto bone, cancer spread to brain, cancer spread to liver, cancer spreadto lung, carcinoid, cervical cancer, choriocarcinoma, chroniclymphocytic leukaemia (CLL), chronic myeloid leukaemia (CML), coloncancer, colorectal cancer, endometrial cancer, eye cancer, gallbladdercancer, gastric cancer, gestational trophoblastic tumours (GTT), hairycell leukaemia, head and neck cancer, hodgkin lymphoma, kidney cancer,laryngeal cancer, leukaemia, liver cancer, lung cancer, lymphoma,melanoma skin cancer, mesothelioma, men's cancer, molar pregnancy, mouthand oropharyngeal cancer, myeloma, nasal and sinus cancers,nasopharyngeal cancer, non hodgkin lymphoma (NHL), oesophageal cancer,ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, rarecancers, rectal cancer, salivary gland cancer, secondary cancers, skincancer (non melanoma), soft tissue sarcoma, stomach cancer, testicularcancer, thyroid cancer, unknown primary cancer, uterine cancer, vaginalcancer, and vulval cancer. Antigen expression, conjugation, loading

In certain embodiments, the polypeptide antigen is expressed within theexogenous antigen-expressing EHC. The polypeptide antigen may beexhibited on the surface of the exogenous antigen-expressing EHC or mayreside within the exogenous antigen-expressing EHC.

In certain embodiments, the polypeptide antigen is conjugated to theexogenous antigen-expressing EHC. The polypeptide antigen usually isconjugated to the surface of the exogenous antigen-expressing EHC.Conjugation may be achieved chemically or enzymatically, by methodsknown in the art and described herein. Non-polypeptide antigens may alsobe conjugated to an exogenous antigen-expressing EHC. In someembodiments, the antigen is not conjugated to the exogenousantigen-expressing EHC.

In certain embodiments, the polypeptide antigen is loaded into theexogenous antigen-expressing EHC. Non-polypeptide antigens may also beloaded within an exogenous antigen-expressing EHC. In some embodiments,the antigen is not loaded into or onto the exogenous antigen-expressingEHC.

In some embodiments, the exogenous antigen-expressing EHC comprises anantigen that is optionally expressed from a recombinant nucleic acid,conjugated to the EHC, loaded into or onto the EHC, and any combinationthereof. Optionally, the exogenous antigen-expressing EHC comprises atherapeutic agent or other payload.

In some embodiments, the exogenous antigen-expressing EHC is generatedby contacting a suitable isolated cell, e.g., an EHC, a reticulaocyte,an EHC precursor, a platelet, or a platelet precursor, with arecombinant nucleic acid encoding an antigen polypeptide. In someembodiments, the antigen polypeptide is encoded by a DNA, which iscontacted with a nucleated erythroid precursor cell or a nucleatedplatelet precursor cell. In some embodiments, the antigen polypeptide isencoded by an RNA, which is contacted with a platelet, a nucleate EHC, anucleated platelet precursor cell, or a reticulocyte. In someembodiments, the antigen is a polypeptide, which is contacted with aprimary platelet, a nucleated EHC, a nucleated platelet precursor cell,a reticulocyte, or an erythrocyte.

A antigen polypeptide may be expressed from a transgene introduced intoan EHC by electroporation, chemical or polymeric transfection, viraltransduction, mechanical membrane disruption, or other method; anantigen polypeptide that is expressed from mRNA that is introduced intoa cell by electroporation, chemical or polymeric transfection, viraltransduction, mechanical membrane disruption, or other method; anantigen polypeptide that is over-expressed from the native locus by theintroduction of an external factor, e.g., a transcriptional activator,transcriptional repressor, or secretory pathway enhancer; and/or anantigen polypeptide that is synthesized, extracted, or produced from aproduction cell or other external system and incorporated into the EHC.

In some embodiments, the antigen is a full-length protein. In someembodiments, the antigen is comprised of one or more polypeptidescontained within the full-length protein, of any length greater thanapproximately 7 amino acids. For example, the polypeptides can be 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 aminoacids, e.g 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 aminoacids. The polypeptides comprising the antigen may comprise one or moreimmunological epitopes which may be conformational epitopes or may belinear epitopes. The antigen may be comprised of one or morepolypeptides from one or more different proteins.

The antigen of interest can be expressed in the circulating cell byfusion to an endogenous cell protein, including but not limited to thoselisted in Table B and Table C. Fusion to an endogenous protein may benecessary because it is thought that during the natural process ofdifferentiation and enucleation, the EHC sheds and discards many of theendogenous proteins required for erythropoiesis but not for matureerythrocyte function such as, e.g. c-Kit (SCF receptor) and transferrin.See e.g. Keerthivasan et al., Stem Cells International 2011; Migliaccio,Haematologica 2010. Proteins that are retained include certain membraneproteins such as, e.g. glycophorin A, band 3, and aquaporin; certaincytoplasmic proteins such as, e.g. hemoglobin alpha, hemoglobin beta,and adenosine deaminase; and cytoskeletal proteins.

The antigen of interest can be expressed in the intracellular space ofthe EHC by a number of methods, including direct expression of thetransgene, fusion to an endogenous intracellular protein such as, e.g.,hemoglobin, fusion to the intracellular domain of endogenous cellsurface proteins such as, e.g. Band 3, glycophorin A, Kell, or fusion toa structural component of the erythroid cytoskeleton.

The antigen of interest can be expressed on the extracellular surface ofthe EHC by a number of methods, including direct expression of thetransgene if it contains a transmembrane domain or other membraneattachment domain, fusion to an endogenous erythroid membrane protein orto the transmembrane domain of said protein such as, e.g. Band 3,glycophorin A, or Kell; or fusion to the GPI-linker acceptor peptide ofan endogenous erythroid GPI-linked cell surface protein such as, e.g.acetylcholinesterase, CD55, CD58 or CD59 (see, e.g. Kooyman et al.,Science 1995).

The antigen of interest can be conjugated to the surface of a culturedEHC by various chemical and enzymatic means, including but not limitedto those listed in Table D, Table D1, and Table E. These methods includechemical conjugation with bifunctional cross-linking agents such as,e.g. an NHS ester-maleimide heterobifunctional crosslinker to connect aprimary amine group with a reduced thiol group. These methods alsoinclude enzymatic strategies such as, e.g. transpeptidase reactionmediated by a sortase enzyme to connect one polypeptide containing theacceptor sequence LPXTG or LPXTA with a polypeptide containing theN-terminal donor sequence GGG, see e.g. Swee et al., PNAS 2013. Themethods also include combination methods, such as e.g. sortase-mediatedconjugation of Click Chemistry handles (an azide and an alkyne) on theantigen and the cell, respectively, followed by a cycloaddition reactionto chemically bond the antigen to the cell, see e.g. Neves et al.,Bioconjugate Chemistry, 2013.

If desired, a catalytic bond-forming polypeptide domain can be expressedon or in an EHC, either intracellularly or extracellularly. Manycatalytic bond-forming polypeptides exist, including transpeptidases,sortases, and isopeptidases, including those derived from Spy0128, aprotein isolated from Streptococcus pyogenes.

It has been demonstrated that splitting the autocatalytic isopeptidebond-forming subunit (CnaB2 domain) of Spy0128 results in two distinctpolypeptides that retain catalytic activity with specificity for eachother. The polypeptides in this system are termed SpyTag and SpyCatcher.Upon mixing, SpyTag and SpyCatcher undergo isopeptide bond formationbetween Asp117 on SpyTag and Lys31 on SpyCatcher (Zakeri and Howarth,JACS 2010, 132:4526). The reaction is compatible with the cellularenvironment and highly specific for protein/peptide conjugation (Zakeri,B.; Fierer, J. O.; Celik, E.; Chittock, E. C.; Schwarz-Linek, U.; Moy,V. T.; Howarth, M. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, E690-E697).SpyTag and SpyCatcher has been shown to direct post-translationaltopological modification in elastin-like protein. For example, placementof SpyTag at the N-terminus and SpyCatcher at the C-terminus directsformation of circular elastin-like proteins (Zhang et al, Journal of theAmerican Chemical Society, 2013).

The components SpyTag and SpyCatcher can be interchanged such that asystem in which molecule A is fused to SpyTag and molecule B is fused toSpyCatcher is functionally equivalent to a system in which molecule A isfused to SpyCatcher and molecule B is fused to SpyTag. For the purposesof this document, when SpyTag and SpyCatcher are used, it is to beunderstood that the complementary molecule could be substituted in itsplace.

A catalytic bond-forming polypeptide, such as a SpyTag/SpyCatchersystem, can be used to attach an exogenous antigen of interest to thesurface of an EHC. The SpyTag polypeptide sequence can be expressed onthe extracellular surface of the EHC. The SpyTag polypeptide can be, forexample, fused to the N terminus of a type-1 or type-3 transmembraneprotein, e.g. glycophorin A, fused to the C terminus of a type-2transmembrane protein, e.g. Kell, inserted in-frame at the extracellularterminus or in an extracellular loop of a multi-pass transmembraneprotein, e.g. Band 3, fused to a GPI-acceptor polypeptide, e.g. CD55 orCD59, fused to a lipid-chain-anchored polypeptide, or fused to aperipheral membrane protein. The nucleic acid sequence encoding theSpyTag fusion can be expressed within an EHC. An exogenous antigen ofinterest can be fused to SpyCatcher. The nucleic acid sequence encodingthe SpyCatcher fusion can be expressed and secreted from the same EHCthat expresses the SpyTag fusion. Alternatively, the nucleic acidsequence encoding the SpyCatcher fusion can be produced exogenously, forexample in a bacterial, fungal, insect, mammalian, or cell-freeproduction system. Upon reaction of the SpyTag and SpyCatcherpolypeptides, a covalent bond will be formed that attaches the exogenousantigen of interest to the surface of the EHC.

In one embodiment, the SpyTag polypeptide may be expressed as a fusionto the N terminus of glycophorin A under the control of the Gatalpromoter in an EHC. An exogenous antigen of interest, for example theexogenous antigens listed in Table F, Table G, Table H, Table I andTable J, fused to the SpyCatcher polypeptide sequence can be expressedunder the control of the Gatal promoter in the same EHC. Upon expressionof both fusion polypeptides, an isopeptide bond will be formed betweenthe SpyTag and SpyCatcher polypeptides, forming a covalent bond betweenthe EHC surface and the exogenous antigen of interest.

In another embodiment, the SpyTag polypeptide may be expressed as afusion to the N terminus of glycophorin A under the control of the Gatalpromoter in an EHC. An exogenous antigen of interest, for example theexogenous antigens listed in Table F, Table G, Table H, Table I andTable J, fused to the SpyCatcher polypeptide sequence can be expressedin a suitable mammalian cell expression system, for example HEK293cells. Upon expression of the SpyTag fusion polypeptide on the EHC, theSpyCatcher fusion polypeptide can be brought in contact with the cell.Under suitable reaction conditions, an isopeptide bond will be formedbetween the SpyTag and SpyCatcher polypeptides, forming a covalent bondbetween the EHC surface and the exogenous antigen of interest.

A catalytic bond-forming polypeptide, such as a SpyTag/SpyCatchersystem, can be used to anchor the exogenous antigen of interest to theintracellular space of an EHC. The SpyTag polypeptide sequence can beexpressed in the intracellular space of the EHC by a number of methods,including direct expression of the transgene, fusion to an endogenousintracellular protein such as, e.g., hemoglobin, fusion to theintracellular domain of endogenous cell surface proteins such as, e.g.Band 3, glycophorin A, Kell, or fusion to a structural component of theerythroid cytoskeleton. The SpyTag sequence is not limited to apolypeptide terminus and may be integrated within the interior sequenceof an endogenous polypeptide such that polypeptide translation andlocalization is not perturbed. An exogenous antigen of interest can befused to SpyCatcher. The nucleic acid sequence encoding the SpyCatcherfusion can be expressed within the same EHC that expresses the SpyTagfusion. Upon reaction of the SpyTag and SpyCatcher polypeptides, acovalent bond will be formed that acts to anchor the antigen of interestin the intracellular space of the EHC.

In one embodiment, an EHC may express SpyTag fused to hemoglobin betaintracellularly. The EHC may be genetically modified with a genesequence that includes a hemoglobin promoter, beta globin gene and aSpyTag sequence such that upon translation, intracellular beta globin isfused to SpyTag at is C terminus. In addition, the EHC expresses a Gatalpromoter-led gene that codes for SpyCatcher driving phenylalaninehydroxylase (PAH) expression such that upon translation, intracellularPAH is fused to SpyCatcher at its N terminus. Upon expression of bothfusion proteins the SpyTag bound beta globin is linked through anisopeptide bond to the SpyCatcher bound PAH in the intracellular space,allowing PAH to be anchored to beta globin and retained duringmaturation.

In another embodiment, the SpyTag polypeptide can be expressed as afusion to the exogenous antigen of interest within an EHC. TheSpyCatcher polypeptide can be expressed as a fusion to the C terminus(intracellular) of glycophorin A within the same EHC. Upon expression ofboth fusion polypeptides, an isopeptide bond will be formed between theSpyTag and SpyCatcher polypeptides, forming a covalent bond between themembrane-anchored endogenous erythroid polypeptide and the exogenousantigen of interest.

In another example, the exogenous antigen of interest may be physicallyloaded into a cultured EHC (as opposed to expressed) by a number ofmethods, including osmotic loading or hypotonic-hypertonic cycling inwhich exogenous antigen diffuses through pores introduced into the EHCmembrane (see e.g. Cremel and Godfrin, Int J Pharm 2013) and fusion to acell penetrating peptide, such as one derived from a bacterial toxin,see e.g. Kwon et al., J Contr Rel 2009.

The exogenous antigen of interest may be expressed from a transgeneintroduced into an EHC by electroporation, chemical or polymerictransfection, viral transduction, mechanical membrane disruption, orother method; an exogenous antigen that is expressed from mRNA that isintroduced into a cell by electroporation, chemical or polymerictransfection, viral transduction, mechanical membrane disruption, orother method; an exogenous antigen polypeptide that is over-expressedfrom the native locus by the introduction of an external factor, e.g. atranscriptional activator, transcriptional repressor, or secretorypathway enhancer; an exogenous antigen that is synthesized, extracted,or produced from a production cell or other external system andincorporated into the EHC.

EHCs of the invention may optionally be loaded with materials (payload)such as peptides, proteins, DNA, RNA, siRNA, and other macromolecules byapplying controlled injury to the cell for a predetermined amount oftime in order to cause perturbations in the cell membrane such that thematerials can be delivered to the inside of the cell (e.g. cytoplasm).

In preferred embodiments, the EHC is a reticulocyte. For example,reticulocytes may be loaded with an mRNA encoding an exogenous antigenby controlled cell injury. The mRNA may be naked or modified, asdesired. mRNA modification that improve mRNA stability and/or decreaseimmunogenicity include, e.g. ARCA: anti-reverse cap analog (m₂^(7,3′-O)GP₃G), GP₃G (Unmethylated Cap Analog), m⁷GP₃G (MonomethylatedCap Analog), m₃ ^(2.2.7)GP₃G (Trimethylated Cap Analog), m5CTP(5′-methyl-cytidine triphosphate), m6ATP(N6-methyl-adenosine-5′-triphosphate), s2UTP (2-thio-uridinetriphosphate), and Ψ (pseudouridine triphosphate).

In another preferred embodiment, the EHC is an erythrocyte. For example,erythrocytes may be loaded with an exogenous antigen by controlled cellinjury. The cell injury can be caused by, for example, pressure inducedby mechanical strain or shear forces, subjecting the cell todeformation, constriction, rapid stretching, rapid compression, or pulseof high shear rate. The controlled cell injury leads to uptake ofmaterial (payload) into the cytoplasm of the cell from the surroundingcell medium.

Using controlled cell injury based on controlled cell deformation (e.g.mechanical deformation of the cell as it passes through theconstriction) leads to uptake of material (payload) by diffusion ratherthan endocytosis. The material (payload) is present in the cytoplasmrather than in endosomes following cellular uptake upon the controlledinjury thereby making the material readily available to the cell.Controlled cell injury, e.g. by controlled deformation, preserves cellviability (e.g. greater than 50%, 70%, or greater than 90%). In certainembodiments, controlled cell injury, e.g. by controlled deformation,preserves the state of cellular differentiation and activity. Ifdesired, a combination treatment is used, e.g., controlled injury bydeformation followed by or preceded by, e.g., electroporation or anothercell membrane permeability increasing method. Optionally, surfactantsmay be used.

Mechanical deformation methods are particularly suitable for cells thatdo not tolerate other membrane permeability increasing methods well,e.g. show decreased viability or a different state of differentiationafter performing such methods. Mechanical deformation methods are alsosuitable for material (payload) that does not tolerate other membranepermeability increasing methods well. Alternatively or in addition, thepayload may not be sufficiently introduced into the cell usingalternative methods, e.g. because of e.g. charge, hydrophobicity, orsize of the payload.

One exemplar method of controlled injury by deformation and devicessuitable for such methods is described, e.g. in PCT Publication No.WO2013059343 INTRACELLULAR DELIVERY, incorporated herein by reference.

In a specific embodiment, a population of reticulocytes is provided thathas been subjected to controlled cell injury by controlled deformation.The cells can, e.g., be compressed and deformed by passage through amicro-channel having a diameter less than that of an individualreticulocyte, thereby causing perturbations in the cell membrane suchthat the membrane becomes porous. Cells are moved, e.g., pushed, throughthe channels or conduits by application of pressure.The compression anddeformation occurs in a delivery medium comprising a payload, e.g. anexogenous polypeptide or oligonucleotide (e.g. DNA, RNA, such as mRNA).For example, the delivery medium may comprise an exogenous antigenlisted in Table F, Table G, Table H, Table I and Table J or coding mRNAthereof. Upon deformation the reticulocyte takes up and retains theexogenous material. Following controlled injury to the cell byconstriction, stretching, and/or a pulse of high shear rate, the cellsare optionally incubated in a delivery medium that contains the material(payload). The cells may be maintained in the delivery medium for a fewminutes to recover, e.g. to close the injury caused by passing throughthe constriction. This may occur at room temperature.

Controlled cell injury as used herein includes: i) virus-mediatedtransfection (e.g. Herpes simplex virus, Adeno virus, Adeno-associatedvirus, Vaccinia virus, or Sindbis virus), ii) chemically-mediatedtransfection, e.g. cationic polymer, calcium phosphate, cationic lipid,polymers, and nanoparticles, such as cyclodextrin, liposomes, cationicliposomes, DEAE-dextran, polyethyleneimine, dendrimer, polybrene,calcium phosphate, lipofectin, DOTAP, lipofectamine, CTAB/DOPE, DOTMA;and iii) physically-mediated transfection, including direct injection,biolistic particle delivery, electroporation, laser-irradiation,sonoporation, magnetic nanoparticles, and controlled deformation (e.g.cell squeezing), as exemplified by micro-needle, nano-needle,femtosyringe, atomic-force microscopy (AFM) tip, gene gun (e.g. goldnanoparticles), Amaxa Nucleofector, phototransfection (multi-photonlaser), impalefection, and magnetofection, and other suitable methodsknown in the art. Any suitable method may be used to obtain the EHCsdescribed herein comprising one or more desired DNA, RNA (e.g. mRNA), orpolypeptides comprising antigen.

Exogenous antigen of interest can be detected on the EHC of theinvention. The presence of the exogenous antigen can be validated andquantified using standard molecular biology methods, e.g. Westernblotting or FACS analysis. Exogenous antigens present in theintracellular environment may be quantified upon cell lysis or usingfluorescent detection.

Manufacturing

In some embodiments, the EHC is generated using a precursorhematopoietic cell, e.g., a CD34+ cell, an erythrocyte, a platelet, amegakaryocyte, or a neutrophil as a source. In some embodiments, theprecursor hematopoietic cell is isolated from a human donor by aGMP-compliant process. In some embodiments, the starting cells aresourced from an autologous donor. In some embodiments, the startingcells are sourced from an allogeneic donor. The donor may be typed forblood cell antigen polymorphisms and/or the donor is genotyped for bloodcell antigens. The donor can be a universal blood donor. In someembodiments, the donor has the Bombay phenotype, .ie.does not expressthe H antigen. In some embodiments, the donor has ABO blood type O andis Rh-negative.

In some embodiments, the EHC is generated using CD34+ hematopoieticprogenitor cells, mobilized peripheral CD34+ cells, or bonemarrow-derived CD34+ cells as a source for the starting material. Insome embodiments, the starting cells are derived from umbilical cordblood, are induced pluripotent stem cells or are embryonic stem cells.

The exogenous antigen-expressing EHC may be cultured. Cultured EHCs canbe scaled up from bench-top scale to bioreactor scale. For example, theEHCs are cultured until they reach saturation density, e.g., 1×10⁵,1×10⁶, 1×10⁷, or greater than 1×10⁷ EHCs per ml. Optionally, uponreaching saturation density, the EHCs can be transferred to a largervolume of fresh medium. The exogenous antigen-expressing EHCs may becultured in a bioreactor, such as, e.g., a Wave-type bioreactor, astirred-tank bioreactor. Various configurations of bioreactors are knownin the art and a suitable configuarion may be chosen as desired.Configurations suitable for culturing and/or expanding populations ofexogenous antigen-expressing EHCs can easily be determined by one ofskill in the art without undue experimentation. The bioreactor can beoxygenated. The bioreactor may optionally contain one or more impellers,a recycle stream, a media inlet stream, and control components toregulate the influx of media and nutrients or to regulate the outflux ofmedia, nutrients, and waste products.

In some embodiments, the bioreactor may contain a population of humanEHCs comprising an exogenous antigen that shed their intracellular DNAover the course of the culture process. For example, the bioreactor maycontain a population of human EHCs, enucleated EHCs, and pyrenocytesafter culture. In a specific embodiment, the human EHCs and enucleatedEHCs comprise an exogenous antigen and the exogenous antigen is retainedby the enucleated EHC, whereas the recombinant nucleic acid encoding theexogenous antigen is not retained by the enucleated cell. In certainembodiments, the enucleated EHC comprising the exogenous antigenexhibits substantially the same osmotic membrane fragility as acorresponding isolated unmodified, uncultured EHC.

In one embodiment. The population of exogenous antigen-expressing EHCsgenerated from EHCs or EHC precursors in the bioreactor undergo a totalexpansion of greater than 20,000-fold in 14 days or greater. In someembodiments, the exogenous antigen is introduced into a cultured orfreshly isolated EHC precursor and after introduction of a recombinantnucleic acid encoding the exogenous antigen the population of exogenousantigen-expressing EHCs generated from the EHC precursors in thebioreactor expands in the bioreactor from the precursor cells by morethan 20,000-fold.

In some embodiments, the bioreactor is a Wave bioreactor or aimpeller-driven agitator. The bioreactor may be aerated by means of asparger. In one embodiment, the bioreactor is disposable. In oneembodiment, the bioreactor is CIP (cleaned in place). The final numberof exogenous antigen-expressing EHCs that may be obtained in abioreactor setting as described herein can be greater than 10⁹, 10¹⁰,10¹¹, 10¹², 10¹³ or greater than 10¹³ EHCs. The density of exogenousantigen-expressing EHCs may be monitored during culture by measuringcell density by hemacytometer counting or by optical density reading at600 nm. Optionally, the culture process is monitored for pH levels,oxygenation, agitation rate, and/or recycle rate.

Processes and Properties

The identity of the exogenous antigen-expressing EHCs can be assessed byin vitro assays. For example, the identity of the exogenousantigen-expressing EHCs is assessed by counting the number of EHCs in apopulation, e.g., by microscopy, by flow cytometry, or by hemacytometry.Alternatively or in addition, the identity of the exogenousantigen-expressing EHCs is assessed by analysis of protein content ofthe EHCs, e.g., by flow cytometry, Western blot, immunoprecipitation,fluorescence spectroscopy, chemiluminescence, mass spectrometry, orabsorbance spectroscopy. In one embodiment, the protein content assayedis a non-surface protein, e.g., an integral membrane protein,hemoglobin, adult hemoglobin, fetal hemoglobin, embryonic hemoglobin, acytoskeletal protein. In one embodiment, the protein content assayed isa surface protein, e.g., a differentiation marker, a receptor, aco-receptor, a transporter, a glycoprotein. In one embodiment, thesurface protein is selected from the list including, but not limited to,glycophorin A, CKIT, transferrin receptor, Band3, Kell, CD45, CD46,CD47, CD55, CD59, CR1. In some embodiments, the identity of theexogenous antigen-expressing EHCs is assessed by analysis of theexogenous antigen content of the EHCs, e.g., by flow cytometry, Westernblot, immunoprecipitation, fluorescence spectroscopy, chemiluminescence,mass spectrometry, or absorbance spectroscopy. For example, the identityof the exogenous antigen-expressing EHCs can be assessed by the mRNAcontent of the EHCs, e.g., by RT-PCR, flow cytometry, or northern blot.The identity of the exogenous antigen-expressing EHCs can be assessed bynuclear material content, e.g., by flow cytometry, microscopy, orsouthern blot, using, e.g., a nuclear stain or a nucleic acid probe.Alternatively or in addition, the identity of the exogenousantigen-expressing EHCs is assessed by lipid content of the EHCs, e.g byflow cytometry, liquid chromatography, or by mass spectrometry.

In some embodiments, the identity of the exogenous antigen-expressingEHCs is assessed by metabolic activity of the EHCs, e.g by massspectrometry, chemiluminescence, fluorescence spectroscopy, absorbancespectroscopy. Metabolic acitivity can be assessed by ATP consumptionrate and/or the metabolic activity is assessed measuring2,3-diphosphoglycerate (2,3-DPG) level in the exogenousantigen-expressing EHC. The metabolic activity can be assessed as therate of metabolism of one of the following, including but not limitedto, Acetylsalicylic acid, N-Acetylcystein, 4-Aminophenol, Azathioprine,Bunolol, Captopril, Chlorpromazine, Dapsone, Daunorubicin,Dehydroepiandrosterone, Didanosin, Dopamine, Epinephrine, Esmolol,Estradiol, Estrone, Etoposide, Haloperidol, Heroin, Insulin,Isoproterenol, Isosorbide dinitrate, LY 217896, 6-mercaptopurine,Misonidazole, Nitroglycerin, Norepinephrine, Para-aminobenzoic acid. Insome embodiments, the identity of the exogenous antigen-expressing EHCsis assessed by partitioning of a substrate by the EHCs, e.g by massspectrometry, chemiluminescence, fluorescence spectroscopy, orabsorbance spectroscopy. The substrate can be one of the following,including but not limited to, Acetazolamide, Arbutine, Bumetamide,Creatinine, Darstine, Desethyldorzolamide, Digoxigenin digitoxoside,Digoxin-16′-glucuronide, Epinephrine, Gentamycin, Hippuric acid,Metformin, Norepinephrine, p-Aminohippuric acid, Papaverine, PenicillinG, Phenol red, Serotonin, Sulfosalicylic acid, Tacrolimus, Tetracycline,Tucaresol, and Vancomycin.

In one embodiment, the population of exogenous antigen-expressing EHCsis differentiated from a precursor cell. In this embodiment, thedifferentiation state of the population of exogenous antigen-expressingEHCs is assessed by an in vitro assay. The in vitro assays include thosedescribed herein for assessing the identity of the EHCs, including butnot limited to expansion rate, number, protein content or expressionlevel, mRNA content or expression level, lipid content, partition of asubstrate, catalytic activity, or metabolic activity.

In some embodiments, the exogenous antigen-expressing EHCs are culturedand the differentiation state of the EHCs is assessed at multiple timepoints over the course of the culture process.

Exogenous antigen-expressing EHCs may be generated using reticulocytesas a source for starting material. The purity of isolated reticulocytesmay be assessed using microscopy in that reticulocytes are characterizedby a reticular (mesh-like) network of ribosomal RNA that becomes visibleunder a microscope with certain stains such as new methylene blue orbrilliant cresyl blue. Surface expression of transferrin receptor (CD71)is also higher on reticulocytes and decreases and they mature toerythrocytes, allowing for enrichment and analysis of reticulocytepopulations using anti-CD71 antibodies (See, e.g., Miltenyi CD71microbeads product insert No. 130-046-201). Alternatively, analysis ofcreatine and hemoglobin A1C content and pyruvate kinase, aspartateaminotransferase, and porphobilinogen deaminase enzyme activity may beused to assess properties of the isolated reticulocytes relative tomature erythrocytes (See, e.g., Brun et al., Blood 76:2397-2403 (1990)).For example, the activity of porphobilinogen deaminase is nearly 9 foldhigher whereas the hemoglobin A1C content is nearly 10 fold less inreticulocytes relative to mature erythrocytes.

In some embodiments, cells suitable for generating exogenousantigen-expressing EHCs are differentiated ex vivo and/or in vivo fromone or more stem cells. In one embodiment, the one or more stem cellsare one or more hematopoietic stem cells.Various assays may be performedto confirm the ex vivo differentiation of cultured hematopoietic stemcells into reticulocytes and erythrocytes, including, for example,microscopy, hematology, flow cytometry, deformability measurements,enzyme activities, and hemoglobin analysis and functional properties(Giarratana et al., Nature Biotech. 23:69-74 (2005)). The phenotype ofcultured hematopoietic stem cells may be assessed using microscopy ofcells stained, for example, with Cresyl Brilliant blue. Reticulocytes,for example, exhibit a reticular network of ribosomal RNA under thesestaining conditions whereas erythrocytes are devoid of staining.Enucleated cells may also be monitored for standard hematologicalvariables including mean corpuscular volume (MCV; femtoliters (fL)),mean corpuscular hemoglobin concentration (MCHC; %) and mean corpuscularhemoglobin (MCH; pg/cell) using, for example, an XE2100 automat (Sysmex,Roche Diagnostics).

In some embodiments, the exogenous antigen-expressing EHCs are assessedfor their basic physical properties, e.g., size, mass, volume, diameter,buoyancy, density, and membrane properties, e.g., viscosity,deformability fluctuation, and fluidity.

In one embodiment, the diameter of the exogenous antigen-expressing EHCsis measured by microscopy or by automated instrumentation, e.g., ahematological analysis instrument. In one embodiment the diameter of theexogenous antigen-expressing EHCs is between about 1-20 microns. In oneembodiment, the diameter of the exogenous antigen-expressing EHCs is atleast in one dimension between about 1-20 microns. In one embodiment,the diameter of the exogenous antigen-expressing EHCs is less than about1 micron. In one embodiment, the diameter of the EHCs in one dimensionis larger than about 20 microns. In one embodiment, the diameter of theexogenous antigen-expressing EHCs is between about 1 micron and about 20microns, between about 2 microns and about 20 microns between about 3microns and about 20 microns between about 4 microns and about 20microns between about 5 microns and about 20 microns between about 6microns and about 20 microns, between about 5 microns and about 15microns or between about 10 microns and about 30 microns.

In one embodiment, the mean corpuscular volume of the exogenousantigen-expressing EHCs is measured using a hematological analysisinstrument. In one embodiment the volume of the mean corpuscular volumeof the EHCs is greater than 10 fL, 20 fL, 30 fL, 40 fL, 50 fL, 60 fL, 70fL, 80 fL, 90 fL, 100 fL, 110 fL, 120 fL, 130 fL, 140 fL, 150 fL, orgreater than 150 fL. In one embodiment the mean corpuscular volume ofthe EHCs is less than 30 fL, 40 fL, 50 fL, 60 fL, 70 fL, 80 fL, 90 fL,100 fL, 110 fL, 120 fL, 130 fL, 140 fL, 150 fL, 160 fL, 170 fL, 180 fL,190 fL, 200 fL, or less than 200 fL. In one embodiment the meancorpuscular volume of the EHCs is between 80-100 femtoliters (fL).

In one embodiment the average buoyant mass of the exogenousantigen-expressing EHCs (pg/cell) is measured using a suspendedmicrochannel resonatory or a double suspended microchannel resonatory(see e.g., Byun et al PNAS 2013 110(19):7580 and Bryan et al. Lab Chip2014 14(3):569).

In one embodiment the dry density of the exogenous antigen-expressingEHCs is measured by buoyant mass in an H2O-D2O exchange assay (see e.g.,Feijo Delgado et al., PLOS One 2013 8(7):e67590).

In some embodiments, the exogenous antigen-expressing EHCs have anaverage membrane deformability fluctuation of standard deviation greaterthan 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 mrad asmeasured by spatial light interference microscopy (SLIM) (see e.g.,Bhaduri et al., Sci Reports 2014, 4:6211).

In one embodiment, the average membrane viscosity of a population ofexogenous antigen-expressing EHCs is measured by detecting the averagefluorescence upon incubation with viscosity-dependent quantum yieldfluorophores (see e.g., Haidekker et al. Chem & Biol 2001 8(2):123).

In one embodiment, the membrane fluidity of the exogenousantigen-expressing EHCs is measured by fluorescence polarization, e.g.,with BMG Labtech POLARstar Omega microplate reader.

For example, to measure deformability reticulocytes may be separatedfrom nucleated cells on day 15 of culture, for example, by passagethrough a deleukocyting filter (e.g., Leucolab LCG2, Macopharma) andsubsequently assayed using ektacytometry. The enucleated cells aresuspended in 4% polyvinylpyrrolidone solution and then exposed to anincreasing osmotic gradient from 60 to 450 mosM. Changes in the laserdiffraction pattern (deformability index) of the cells are recorded as afunction of osmolarity, to assess the dynamic deformability of the cellmembrane. The maximum deformability index achieved at a physiologicallyrelevant osmolarity is related to the mean surface area of erythrocytes.

In some embodiments, the exogenous antigen-expressing EHCs are analyzedfor hemoglobin contents. Assays of hemoglobin may be used to assess thephenotype of differentiated cells (Giarratana et al., Nature Biotech.23:69-74 (2005)). For example, high performance liquid chromatography(HPLC) using a Bio-Rad Variant II Hb analyzer (Bio-Rad Laboratories) maybe used to assess the percentage of various hemoglobin fractions. Oxygenequilibrium may be measured using a continuous method with adouble-wavelength spectrophotometer (e.g., Hemox analyzer, TCS). Thebinding properties of hemoglobin may be assessed using flash photolysis.In this method, the rebinding of CO to intracellular hemoglobintetramers are analyzed at 436 nm after photolysis with a 10 nanosecondpulse at 532 nm.

The exogenous antigen-expressing EHCs described herein can be purifiedfollowing manufacture if desired. Many suitable methods of purificationare known in the art. For example, the exogenous antigen-expressing EHCsare purified by centrifugation, magnetophoresis, irradiation,acoustophoresis, and chemical or physical enucleation. In one embodimentexogenous antigen-expressing EHCs are purified by ex vivo maturationwith, e.g., a stromal cell co-culture. In one embodiment, exogenousantigen-expressing EHCs are purified by chemical or enzymatic treatmentof EHCs, e.g by treatment with a deglycosylation enzyme.

In one embodiment the exogenous antigen-expressing EHCs are purified bydisabling any residual replicative potential of the exogenousantigen-expressing EHCs. In one embodiment the exogenousantigen-expressing EHCs are subjected to radiation, e.g., X rays, gammarays, beta particles, alpha particles, neutrons, protons, elementalnuclei, UV rays in order to damage residual replication-competentnucleic acids.

Ionizing radiation is energy transmitted via X rays, gamma rays, betaparticles (high-speed electrons), alpha particles (the nucleus of thehelium atom), neutrons, protons, and other heavy ions such as the nucleiof argon, nitrogen, carbon, and other elements. X rays and gamma raysare electromagnetic waves like light, but their energy is much higherthan that of light (their wavelengths are much shorter). Ultraviolet(UV) light is a radiation of intermediate energy that can damage cellsbut UV light differs from the forms of electromagnetic radiationmentioned above in that it does not cause ionization (loss of anelectron) in atoms or molecules, but rather excitation (change in energylevel of an electron). The other forms of radiation—particles—are eithernegatively charged (electrons), positively charged (protons, alpha rays,and other heavy ions), or electrically neutral (neutrons).

Radiation-induced ionizations may act directly on the cellular componentmolecules or indirectly on water molecules, causing water-derivedradicals. Radicals react with nearby molecules in a very short time,resulting in breakage of chemical bonds or oxidation (addition of oxygenatoms) of the affected molecules. The major effect in cells is DNAbreaks. Since DNA consists of a pair of complementary double strands,breaks of either a single strand or both strands can occur. However, thelatter is believed to be much more important biologically. Mostsingle-strand breaks can be repaired normally thanks to thedouble-stranded nature of the DNA molecule (the two strands complementeach other, so that an intact strand can serve as a template for repairof its damaged, opposite strand). In the case of double-strand breaks,however, repair is more difficult and erroneous rejoining of broken endsmay occur. These so-called misrepairs result in induction of mutations,chromosome aberrations, or cell death.

Deletion of DNA segments is the predominant form of radiation damage incells that survive irradiation. It may be caused by (1) misrepair of twoseparate double-strand breaks in a DNA molecule with joining of the twoouter ends and loss of the fragment between the breaks or (2) theprocess of cleaning (enzyme digestion of nucleotides—the componentmolecules of DNA) of the broken ends before rejoining to repair onedouble-strand break.

Radiations differ not only by their constituents (electrons, protons,neutrons, etc.) but also by their energy. Radiations that cause denseionization along their track (such as neutrons) are calledhigh-linear-energy-transfer (high-LET) radiation, a physical parameterto describe average energy released per unit length of the track. (Seethe accompanying figure.) Low-LET radiations produce ionizations onlysparsely along their track and, hence, almost homogeneously within acell. Radiation dose is the amount of energy per unit of biologicalmaterial (e.g., number of ionizations per cell). Thus, high-LETradiations are more destructive to biological material than low-LETradiations—such as X and gamma rays—because at the same dose, thelow-LET radiations induce the same number of radicals more sparselywithin a cell, whereas the high-LET radiations—such as neutrons andalpha particles—transfer most of their energy to a small region of thecell. The localized DNA damage caused by dense ionizations from high-LETradiations is more difficult to repair than the diffuse DNA damagecaused by the sparse ionizations from low-LET radiations.

In one embodiment, a population of exogenous antigen-expressing EHCs aresubjected to gamma irradiation using an irradiation dose of more than 1,5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, or more than 100kGy.

In one embodiment, a population of exogenous antigen-expressing EHCs aresubjected to X-ray irradiation using an irradiation dose of more than0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000,6000, 7000, 8000, 9000, 10000, or greater than 10000 mSv.

The purity of a population of exogenous antigen-expressing EHCs can beassessed by measuring the homogeneity of the population. In oneembodiment, the average distribution width of the exogenousantigen-expressing EHCs is measured by a hematological analysisinstrument. In one embodiment, the population of exogenousantigen-expressing EHCs has a reticulocyte to non-reticulocyte ratiogreater than 10, 100, 1000, 10⁴, 10⁵, 10⁶, or greater than 10⁶. Thehomogeneity of the population of exogenous antigen-expressing EHCs maybe assessed by measuring the stromal cell content of the population. Inone embodiment, the population of exogenous antigen-expressing EHCs hasless than 1 ppb of stromal cells. Alternatively or in addition, thehomogeneity of the population of exogenous antigen-expressing EHCs isassessed by measuring the viral titer and/or a bacterial colony formingpotential of the population.

In one embodiment the homogeneity of a population of exogenousantigen-expressing EHCs is assessed by an in vitro assay. The in vitroassays include those described herein for assessing the identity of theEHCs, including but not limited to expansion rate, number, proteincontent or expression level, mRNA content or expression level, lipidcontent, partition of a substrate, catalytic activity, or metabolicactivity.

Mature erythrocytes for use in generating the exogenousantigen-expressing EHCs may be isolated using various methods such as,for example, a cell washer, a continuous flow cell separator, densitygradient separation, fluorescence-activated cell sorting (FACS),Miltenyi immunomagnetic depletion (MACS), or a combination of thesemethods (See, e.g., van der Berg et al., Clin. Chem. 33:1081-1082(1987); Bar-Zvi et al., J. Biol. Chem. 262:17719-17723 (1987); Goodmanet al., Exp. Biol. Med. 232:1470-1476 (2007)).

Erythrocytes may be isolated from whole blood by simple centrifugation(See, e.g., van der Berg et al., Clin. Chem. 33:1081-1082 (1987)). Forexample, EDTA-anticoagulated whole blood may be centrifuged at 800×g for10 mM at 4° C. The platelet-rich plasma and buffy coat are removed andthe red blood cells are washed three times with isotonic saline solution(NaCl, 9 g/L).

Alternatively, erythrocytes may be isolated using density gradientcentrifugation with various separation mediums such as, for example,Ficoll, Hypaque, Histopaque, Percoll, Sigmacell, or combinationsthereof. For example, a volume of Histopaque-1077 is layered on top ofan equal volume of Histopaque-1119. EDTA-anticoagulated whole blooddiluted 1:1 in an equal volume of isotonic saline solution (NaCl, 9 g/L)is layered on top of the Histopaque and the sample is centrifuged at700×g for 30 min at room temperature. Under these conditions,granulocytes migrate to the 1077/1119 interface, lymphocytes, othermononuclear cells and platelets remain at the plasma/1077 interface, andthe red blood cells are pelleted. The red blood cells are washed twicewith isotonic saline solution.

Alternatively, erythrocytes may be isolated by centrifugation using aPercoll step gradient (See, e.g., Bar-Zvi et al., J. Biol. Chem.262:17719-17723 (1987)). For example, fresh blood is mixed with ananticoagulant solution containing 75 mM sodium citrate and 38 mM citricacid and the cells washed briefly in Hepes-buffered saline. Leukocytesand platelets are removed by adsorption with a mixture of α-celluloseand Sigmacell (1:1). The erythrocytes are further isolated fromreticulocytes and residual white blood cells by centrifugation through a45/75% Percoll step gradient for 10 mM at 2500 rpm in a Sorvall SS34rotor. The erythrocytes are recovered in the pellet while reticulocytesband at the 45/75% interface and the remaining white blood cells band atthe 0/45% interface. The Percoll is removed from the erythrocytes byseveral washes in Hepes-buffered saline. Other materials that may beused to generate density gradients for isolation of erythrocytes includeOptiPrep™, a 60% solution of iodixanol in water (from Axis-Shield,Dundee, Scotland).

Erythrocytes may be separated from reticulocytes, for example, usingflow cytometry (See, e.g., Goodman el al., Exp. Biol. Med. 232:1470-1476(2007)). In this instance, whole blood is centrifuged (550×g, 20 min,25° C.) to separate cells from plasma. The cell pellet is resuspended inphosphate buffered saline solution and further fractionated onFicoll-Paque (1.077 density), for example, by centrifugation (400×g, 30min, 25° C.) to separate the erythrocytes from the white blood cells.The resulting cell pellet is resuspended in RPMI supplemented with 10%fetal bovine serum and sorted on a FACS instrument such as, for example,a Becton Dickinson FACSCalibur (BD Biosciences, Franklin Lakes, N.J.,USA) based on size and granularity.

Erythrocytes may be isolated by immunomagnetic depletion (See, e.g.,Goodman, el al., (2007) Exp. Biol. Med. 232:1470-1476). In thisinstance, magnetic beads with cell-type specific antibodies are used toeliminate non-erythrocytes. For example, erythrocytes are isolated fromthe majority of other blood components using a density gradient asdescribed herein followed by immunomagnetic depletion of any residualreticulocytes. The cells are pre-treated with human antibody serum for20 min at 25° C. and then treated with antibodies against reticulocytespecific antigens such as, for example, CD71 and CD36. The antibodiesmay be directly attached to magnetic beads or conjugated to PE, forexample, to which magnetic beads with anti-PE antibody will react. Theantibody-magnetic bead complex is able to selectively extract residualreticulocytes, for example, from the erythrocyte population.

Erythrocytes may also be isolated using apheresis. The process ofapheresis involves removal of whole blood from a patient or donor,separation of blood components using centrifugation or cell sorting,withdrawal of one or more of the separated portions, and transfusion ofremaining components back into the patient or donor. A number ofinstruments are currently in use for this purpose such as for examplethe Amicus and Alyx instruments from Baxter (Deerfield, Ill., USA), theTrima Accel instrument from Gambro BCT (Lakewood, Colo., USA), and theMCS+9000 instrument from Haemonetics (Braintree, Mass., USA). Additionalpurification methods may be necessary to achieve the appropriate degreeof cell purity.

In some embodiments, the exogenous antigen-expressing EHCs aredifferentiated ex vivo and/or in vivo from one or more reticulocytes.Reticulocytes may be used to generate exogenous antigen-expressing EHCs.Reticulocytes are immature red blood cells and compose approximately 1%of the red blood cells in the human body. Reticulocytes develop andmature in the bone marrow. Once released into circulation, reticulocytesrapidly undergo terminal differentiation to mature erythrocytes. Likemature erythrocytes, reticulocytes do not have a cell nucleus. Unlikemature erythrocytes, reticulocytes maintain the ability to performprotein synthesis. In some embodiments, recombinant nucleic acid (suchas mRNA) encoding an exogenous antigen is introduced into reticulocytesto generate exogenous antigen-expressing EHCs.

Reticulocytes of varying age may be isolated from peripheral blood basedon the differences in cell density as the reticulocytes mature.Reticulocytes may be isolated from peripheral blood using differentialcentrifugation through various density gradients. For example, Percollgradients may be used to isolate reticulocytes (See, e.g., Noble el al.,Blood 74:475-481 (1989)). Sterile isotonic Percoll solutions of density1.096 and 1.058 g/ml are made by diluting Percoll (Sigma-Aldrich, SaintLouis, Mo., USA) to a final concentration of 10 mM triethanolamine, 117mM NaCl, 5 mM glucose, and 1.5 mg/ml bovine serum albumin (BSA). Thesesolutions have an osmolarity between 295 and 310 mOsm. Five milliliters,for example, of the first Percoll solution (density 1.096) is added to asterile 15 ml conical centrifuge tube. Two milliliters, for example, ofthe second Percoll solution (density 1.058) is layered over the higherdensity first Percoll solution. Two to four milliliters of whole bloodare layered on top of the tube. The tube is centrifuged at 250×g for 30min in a refrigerated centrifuge with swing-out tube holders.Reticulocytes and some white cells migrate to the interface between thetwo Percoll layers. The cells at the interface are transferred to a newtube and washed twice with phosphate buffered saline (PBS) with 5 mMglucose, 0.03 mM sodium azide and 1 mg/ml BSA. Residual white bloodcells are removed by chromatography in PBS over a size exclusion column.

Alternatively, reticulocytes may be isolated by positive selection usingan immunomagnetic separation approach (See, e.g., Brun et al., Blood76:2397-2403 (1990)). This approach takes advantage of the large numberof transferrin receptors that are expressed on the surface ofreticulocytes relative to erythrocytes prior to maturation. Magneticbeads coated with an antibody to the transferrin receptor may be used toselectively isolate reticulocytes from a mixed blood cell population.Antibodies to the transferrin receptor of a variety of mammalianspecies, including human, are available from commercial sources (e.g.,Affinity BioReagents, Golden, Colo., USA; Sigma-Aldrich, Saint Louis,Mo., USA). The transferrin antibody may be directly linked to themagnetic beads. Alternatively, the transferrin antibody may beindirectly linked to the magnetic beads via a secondary antibody. Forexample, mouse monoclonal antibody 10D2 (Affinity BioReagents, Golden,Colo., USA) against human transferrin may be mixed with immunomagneticbeads coated with a sheep anti-mouse immunoglobulin G (Dynal/Invitrogen,Carlsbad, Calif., USA). The immunomagnetic beads are then incubated witha leukocyte-depleted red blood cell fraction. The beads and red bloodcells are incubated at 22° C. with gentle mixing for 60-90 min followedby isolation of the beads with attached reticulocytes using a magneticfield. The isolated reticulocytes may be removed from the magnetic beadsusing, for example, DETACHaBEAD® solution (from Invitrogen, Carlsbad,Calif., USA). Alternatively, reticulocytes may be isolated from in vitrogrowth and maturation of CD34+ hematopoietic stem cells using themethods described herein.

Terminally-differentiated, enucleated erythrocytes can be separated fromother cells based on their DNA content. In a non-limiting example, cellsare first labeled with a vital DNA dye, such as Hoechst 33342(Invitrogen Corp.). Hoechst 33342 is a cell-permeant nuclearcounterstain that emits blue fluorescence when bound to double-strandedDNA. Undifferentiated precursor cells, macrophages or other nucleatedcells in the culture are stained by Hoechst 33342, while enucleatederythrocytes are Hoechst-negative. The Hoechst-positive cells can beseparated from enucleated erythrocytes by using fluorescence activatedcell sorters or other cell sorting techniques. The Hoechst dye can beremoved from the isolated erythrocytes by dialysis or other suitablemethods.

A population of exogenous antigen-expressing EHCs can be purified byreducing the nuclear material content of the population of EHCs. Forexample, the enucleation rate of the population of EHCs is increased,and/or the number of enucleated exogenous antigen-expressing EHCs isincreased or enriched.

Populations of exogenous antigen-expressing EHCs can be incubated with asmall molecule, e.g., an actin inhibitor, e.g., cytochalasin A, B, C, D,E, F, H, J, and then centrifuged to remove nuclear material.Alternatively or in addition, a population of exogenousantigen-expressing EHCs can be mechanically manipulated by passingthrough progressively smaller size-restrictive filters to remove nuclearmaterial. The population of exogenous antigen-expressing EHCs may alsobe incubated on a fibronectin-coated plastic surface to increase theremoval of nuclear material. In one embodiment, the population ofexogenous antigen-expressing EHCs is incubated in co-culture withstromal cells, e.g., macrophages, to increase the removal of nuclearmaterial.

In some embodiments, the population of exogenous antigen-expressing EHCsis greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or greaterthan 99.9% enucleated.

In some embodiments, the exogenous antigen-expressing EHCs are notco-cultured with support cells, e.g., with an adherent stromal layer. Insome embodiments, the population of exogenous antigen-expressing EHCs isgenerated by contacting EHCs with an exogenous antigen anddifferentiating the EHCs to obtain a population of enucleated cellscomprising the exogenous antigen. The population of exogenousantigen-expressing EHCs is obtained without an enrichment step, such asgravitational separation, magnetic or fluorescent sorting, irradiation,poisoning of nucleated cells, and the like to select for enucleatedcells.

In some embodiments, the population of exogenous antigen-expressing EHCsis comprised of greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, orgreater than 99.9% of exogenous antigen-expressing EHCs that lacknuclear material as assessed by an assay to detect nuclear material suchas those described herein.

In some embodiments, the presence, biological activity and/or functionof an exogenous antigen, such as an exogenous antigen polypeptideexhibited by exogenous antigen-expressing EHCs is assessed. Manysuitable assays are available and known in the art.

In one embodiment, the exogenous antigen is a polypeptide on the surfaceof the exogenous antigen-expressing EHC. The presence of the exogenousantigen can be assessed by assays including but not limited to flowcytometry, western blotting, RT-PCR, Northern blotting, Coombsrosetting, mass spectrometry. In one embodiment, the exogenous antigenis a polypeptide in the interior of the exogenous antigen-expressingEHC. The presence of the exogenous antigen can be assessed by assaysincluding but not limited to Western blotting, RT-PCR, Norther blotting,PCR, Southern blotting, mass spectrometry.

In one embodiment, the exogenous antigen is a nucleic acid on thesurface of the exogenous antigen-expressing EHC. The presence of theexogenous antigen can be assessed by assays including but not limited toflow cytometry, flow cytometry with a homologous fluorescent probe,southern blotting, northern blotting, PCR. In one embodiment, theexogenous antigen is a nucleic acid in the interior of the exogenousantigen-expressing EHC. The presence of the exogenous antigen can beassessed by assays including but not limited to southern blotting,northern blotting, PCR.

In one embodiment, the exogenous antigen is a small molecule on thesurface of the exogenous antigen-expressing EHC. The presence of theexogenous antigen can be assessed by assays including but not limited toflow cytometry, mass spectrometry. In one embodiment, the exogenousantigen is a small molecule in the interior of the exogenousantigen-expressing EHC. The presence of the exogenous antigen can beassessed by assays including but not limited to nass spectrometry,fluorescence spectroscopy.

In one embodiment, the exogenous antigen is a lipid in the membrane ofthe exogenous antigen-expressing EHC. The presence of the exogenousantigen can be assessed by assays including but not limited to massspectrometry, flow cytometry, membrane solubility, fluorescencepolarization, spatial light interferences microscopy.

In one embodiment, the exogenous antigen is fluorescent or is fused to afluoresecent molecule or is co-expressed from a recombinant nucleic acid(e.g., in a vector) with a fluorescent reporter protein like GFP. Thepresence of the exogenous antigen in or on the exogenousantigen-expressing EHC can be assessed by assays including but notlimited to flow cytometry, fluorescence spectroscopy, absorbancespectroscopy.

In one embodiment, the exogenous antigen is a gaseous molecule. Thepresence of the exogenous antigen in or on the exogenousantigen-expressing EHC can be assessed by assays including but notlimited to chemiluminescence assays, mass spectroscopy.

The presence of the exogenous antigen in or on the exogenousantigen-expressing EHC can be assessed by flow cytometry in aquantitative fashion using calibration beads such as commerciallyavailable cytometry calibration beads to quantify the number ofexogenous antigens on an individual EHC. Alternatively or in addition,the presence of the exogenous antigen in or on the exogenousantigen-expressing EHC can be assessed by Western blot in a quantitativefashion using a standard of known concentration that is detectable usingthe same detection reagents as the exogenous antigen, and in this waythe number of exogenous antigens on an individual EHC can be quantified.

In some embodiments, the presence of two or more different exogenousantigens can be assessed by the same or different methods, eithersimultaneously, in sequential fashion, or in parallel. For example, inone embodiment an exogenous antigen on the surface can be assessed byflow cytometry using an antibody specific to the exogenous antigen and adifferent exogenous antigen not on the surface that is fluoresecent canbe assessed by fluorescent signal using a different channel in flowcytometry. In a different example, an exogenous antigen on the surfacecan be assessed by flow cytometry and a different exogenous antigen noton the surface can be assessed by Western blot.

In a specific embodiment, the exogenous antigen is retained on theexogenous antigen-expressing EHC following terminal differentiation ofthe cell source. For example, the exogenous antigen-expressing EHC isgenerated from a cultured EHC and the expression or presence of theexogenous antigen is assessed following terminal differentiation of thecell by a suitable method, e.g., by flow cytometry, Western blot,immunoprecipitation, fluorescence spectroscopy, chemiluminescence,Southern blot, Northern blot, or absorbance spectroscopy.

In a specific embodiment, the exogenous antigen is retained on theexogenous antigen-expressing EHC following circulation in vivo afteradministration of the exogenous antigen-expressing EHC to a subject. Theexogenous antigen-expressing EHC can be injected into a laboratoryanimal or animal model, such as a mouse intravenously, e.g., via thetail vein, or is injected into a human intravenously. Then blood isdrawn and the presence of the exogenous antigen on the exogenousantigen-expressing EHC is assessed by suitable assay, e.g., by flowcytometry, Western blot, immunoprecipitation, fluorescence spectroscopy,chemiluminescence, Southern blot, Northern blot, or absorbancespectroscopy.

In some embodiments, the biological activity of the exogenous antigen inor on the exogenous antigen-expressing EHC, the overall biologicalactivity of the EHC, and the overall activity of a population of EHCscan be assessed by in vitro assays.

In some embodiments, the activity of the exogenous antigen-expressingEHC is rapidly iterated using a model cell line. For example, a libraryof suitable exogenous antigens is expressed in a model cell line, e.g.,HEK293T or K562, and the activity is assessed via a suitable assay; thenthe best exogenous antigen candidate, e.g., the one that is expressed atthe highest level or one that demonstrates the highest activity in thesuitable assay, is expressed, e.g., in cultured EHCs to generateexogenous antigen-expressing EHCs.

In one embodiment, the activity of the exogenous antigen-expressing EHCis rapidly iterated using a cultured mouse model. For example, a libraryof suitable exogenous antigens is expressed in cultured mouse EHCs;activity is assessed in a suitable mouse model of disease or a suitablemouse model system for assessing activity; the best exogenous antigencandidate, e.g., the one that is expressed at the highest level or theone that demonstrates the highest activity in the suitable assay, isthen expressed, e.g., in cultured EHCs to generate exogenousantigen-expressing EHC.

In some instances, the exogenous antigen is an enzyme and the activityof the exogenous antigen can be assessed by an enzymatic assay in whichthe disappearance of a specific substrate molecule is detected or theappearance of a specific product molecule is detected. Such assaysinclude but are not limited to, colorimetric assays, mass spectrometry,HPLC, fluorescent assays.

For example, a) the exogenous antigen is adenosine deaminase (ADA) andthe enzymatic assay detects the conversion of adenosine to inosine; b)the exogenous antigen is phenylalanine hydroxylase (PAH) and the assaydetects the conversion of phenylalanine to tyrosine; c) the exogenousantigen is phenylalanine ammonia lyase (PAL) and the assay detects theconversion of phenylalanine to trans-cinnamic acid; d) the exogenousantigen is thymidine phosphorylase (TP) and the assay detects theconversion of thymidine to thymine and 2-deoxy-ribose; e) the exogenousantigen is Purine nucleoside phosphorylase (PNP) and the assay detectsthe conversion of inosine to hypoxanthine, adenosine into adenine, andguanosine into guanine; f) the exogenous antigen is homogentisate1,2-dioxygenase (HDG) and the assay detects the conversion ofhomogentisate to maleylacetoacetate; g) the exogenous antigen iscystathionine beta synthase and the assay detects the conversion ofserine and homocysteine to cystathionine; h) the exogenous antigen isoxalate oxidase and the assay detects the oxidation of oxalate.

In some embodiments, activity of the exogenous antigen-expressing EHC isassessed in an animal model, for example a mouse model, andimmunodeficient mouse, or an NSG mouse, of a disease, for example ametabolic disease or an enzyme deficiency, or that can demonstrate theeffect of the exogenous antigen-expressing EHC, for example a mouse intowhich a substrate is injected and the product of the exogenousantigen-mediated conversion measured.

In one embodiment, the exogenous antigen is complement receptor 1 (CR1)polypeptide, a derivative or functional fragment thereof. The activityof the CR1 exogenous antigen can be assessed in several ways including,for example, the specific capture of immune complexes by the CR1exogenous antigen, the efficient transfer of the immune complexes tomacrophages, or the in vivo clearance of immune complexes from a mouse.

Functionality of EHCs overexpressing CR1 exogenous antigen may beassessed by one or more processes: capture of immune complexes on theEHC surface comprising CR1 exogenous antigen, release of the immunecomplexes to macrophages while sparing the EHC comprising CR1 exogenousantigen, and proper circulation of the EHCs comprising CR1 exogenousantigen. These three parameters can be assayed in vitro Immune complexcapture assays are described in the art, e.g., Oudin et al., J Immunol2000 and Schifferli et al., J Immunol 1991. For example, labeled immunecomplexes are incubated with EHCs expressing native CR1 or CR1 exogenousantigen polypeptide or a fragment thereof and the number of immunecomplexes captured by the EHCs is assayed by flow cytometry. Macrophagetransfer assays are described in the art, e.g., Kuhn et al., J Immunol1998. For example, labeled immune complexes loaded onto erythrocytesexpressing native CR1 or CR1 exogenous antigen polypeptide or a fragmentthereof are incubated with macrophages. The transfer of immune complexfrom erythrocyte surface to macrophage, and the consumption or sparingof erythrocytes by macrophages, can be measured by flow cytometry.Proper circulation can be predicted by analyzing EHC morphology anddeformability. Morphology of EHCs expressing native CR1 or CR1 exogenousantigen polypeptide or a fragment thereof can be assessed by eye usingstandard microscopy techniques, as described e.g., by Giarratana et al.,Blood 2011 and Repik et al., Clin Exp Immunol 2005. Deformability ofEHCs expressing native CR1 or CR1 exogenous antigen polypeptide or afragment thereof can be assessed by ektacytometry, also known aslaser-assisted optical rotational cell analysis (LORCA), as describede.g., Giarratana et al., Blood 2011.

For example, an exogenous CR1 antigen-expressing EHC (the EHC comprisesa CR1 polypeptide exogenous antigen) is incubated with immune complexes,such as in vivtro generated immune complexes or patient-derived immunecomplexes. The capture of the immune complexes by the CR1 exogenousantigen is assessed by, for example, flow cytometry using a fluorescentmarker in the immune complex or by flow cytometry using a secondarydetection agent against an element of the immune complex.

In one embodiment, the exogenous CR1 antigen-expressing EHC is firstincubated wih immune complexes and then incubated with macrophages, suchas primary macrophages, primary monocytes, cultured macrophages,cultured monocytpes, U937 cells, PMA-activated U937 cells, AA9 cells,RAW 264.7 cells, J774 Cells, THP1 cells, KG-1 cells, NR8383 cells,MV-4-11 cells, 3D4/31 cells, MD cells, Fcwf-4 cells, DH82 cells. Themacrophages are assayed by, for example, flow cytometry or radiography,for the presence of immune complexes transferred by the exogenous CR1antigen-expressing EHC. The transfer of captured immune complexes fromcultured EHCs to macrophages is a standard assay in the art, see forexample: Repik et al. 2005 Clin Exp Immunol. 140:230; Li et al. 2010Infection Immunity 78(7):3129.

In one embodiment, activity of the exogenous CR1 antigen-expressing EHCis assessed in an animal model. For example, a suitable mouse model maybe used, such as an immunodeficient mouse, or an NSG mouse. The mousedisease model can be for example an immune complex disease, such aslupus. Mouse models include NZBWF1/J, MRL/MpJ, MRL/MpJ-Fas(lpr),Smn.C3-Fasl/J, NZM2410/Aeg, 129S4-Cd48, Cg-Sle1, NZM-Sle1 Sle2Sle3/LmoJ, and BXSB.129P2. Alternatively or in addition, a diseasephenotype may be introduced into a mouse, e.g., by injection of immunecomplexes. The exogenous CR1 antigen-expressing EHCs may be injectedinto any suitable mouse (or other animal model) to test one or morebiological effects of the EHC, e.g., the clearance of the injectedimmune complexes by the exogenous CR1 antigen-expressing EHC.

In one embodiment, the exogenous antigen is a complement regulatorymolecule or has complement regulatory activity. This activity of theexogenous antigen can be assessed by both in vitro and in vivo assays.For instance, the activity of the exogenous antigen can be assessed bymeasuring the reduction in an in vitro complement activation assay,e.g., CH50 assay that measures complement-mediated lysis of sensitizedsheep erythroctyes, or AH50 assay that measured alternate pathwaycomplement-mediated lysis of non-sensitized rabbit erythrocytes.Alternatively, the activity of the exogenous antigen can be assessed bydetecting the cleavage or absence of cleavage, which may or may notexpose a neoepitope, of a recombinant complement component that has beenincubated with the exogenous antigen, including but not limited to e.g.,the cleavage of recombinant C2 into C2a and C2b, the cleavage of factorB into factor Ba and factor Bb, the cleavage of factor C3b into iC3bHand iC3bL, the cleavage of C3bBb into C3b and Bb, the cleavage of C4bBbinto C4b and Bb, or the cleavage of factor C4b into iC4bH and iC4bL. Thecleavage or absence of cleavage of a suitable recombinant complementcomponent can be assessed by protein analysis methods known in the artincluding, but not limited to, e.g., chromatography, gelelectrophoresis, ELISA, and western blotting. Suitable in vivo assaysfor exogenous antigen activity include injection of the exogenousantigen-expressing EHC into animal, for example a mouse, and examiningthe deposition of complement factors, for example membrane attackcomplex, by histological staining

In one embodiment, the exogenous antigen is capable of binding orcapturing a target and the activity of the exogenous antigen can beassessed by detecting the captured target on the exogenous antigen invitro or in vivo.

In one embodiment, the exogenous antigen-expressing EHC is incubatedwith the target in vitro, and the capture of the target by the exogenousantigen is detectected using an in vitro assay including but not limitedto, for example, flow cytometry, immunohistochemistry, magneticseparation, radiography, colony-forming assays, microscopy.

In one embodiment, the exogenous antigen-expressing EHC is incubatedwith the target in vitro, and the capture of the target by the exogenousantigen is detected using an in vitro co-culture assay including but notlimited to for example a macrophage consumption assay of opsonizedexogenous antigen-expressing EHC, a T cell activation assay, a B cellstimulation assay, a mast cell degranulation assay, an infectiouspotential assay.

In an embodiment, the exogenous antigen-expressing EHC is incubated withthe target in vitro, and the release or off-rate of the captured targetis measured using an in vitro assay including but not limited to, forexample, flow cytometry, immunohistochemistry, magnetic separation,radiography, colony-forming assays, microscopy.

The capture of the target by the exogenous antigen-expressing EHC can beassayed in an in vivo assay, for example in an animal, including a mousemodel of diseases in which the target is naturally present in the mouse.Suitable diseases include bacterial infections, viral infections, fungalinfections, immune complex diseases, self-antibody diseases,hyperlipidemia, hyperglycemia. In other mouse models, the target isadministered to the mouse externally, e.g., by injection or by feeding.In these assays, the capture of the target by the exogenousantigen-expressing EHC is assayed either by examining the animal, e.gthe plasma, the tissue, for reduction or retention of the target, or byisolating or collecting the exogenous antigen-expressing EHC from theanimal, e.g., from the blood, from the plasma, from a tissue, andassaying the presence of the target on the exogenous antigen using an invitro assay including, but not limited to, for example, flow cytometry,immunohistochemistry, magnetic separation, radiography, colony-formingassays, microscopy.

In some embodiments, the exogenous antigen is capable of binding orcapturing a target and substantially increasing the clearance of thetarget in vivo, or substantially reducing the concentration of thetarget in circulation. The activity of the exogenous antigen on theexogenous antigen-expressing EHC can be assessed by detecting theenhanced clearance of the target in vitro or in vivo.

In one embodiment, the exogenous antigen-expressing EHC is incubatedwith the target in vitro, and the capture of the target by the exogenousantigen is detectected using an in vitro assay including but not limitedto, for example, flow cytometry, immunohistochemistry, magneticseparation, radiography, colony-forming assays, microscopy.Subsequently, the exogenous antigen-expressing EHC is incubated in aco-culture assay with a cell known to promote clearance, for example amacrophage or a monocyte, and the clearance of the target and exogenousantigen-expressing EHC is assessed by, for example, flow cytometry,immunohistochemistry, magnetic separation, radiography, colony-formingassays, microscopy.

In one embodiment, the exogenous antigen-expressing EHC is incubatedwith the target in vitro, and the capture of the target by the exogenousantigen is detectected using an in vitro assay including but not limitedto, for example, flow cytometry, immunohistochemistry, magneticseparation, radiography, colony-forming assays, microscopy.Subsequently, the exogenous antigen-expressing EHC is incubated in aphysical system that mimics the clearance mechanism of the EHC in vivo,for example an artificial spleen, a microchannel, a packed column, aresin, a tissue explant, a centrifuge, and the clearance of the targetand exogenous antigen-expressing EHC is assessed by, for example, flowcytometry, immunohistochemistry, magnetic separation, radiography,colony-forming assays, microscopy.

In one embodiment, the clearance of the target by the exogenousantigen-expressing EHC is assayed in an in vivo assay, for example in ananimal, including, for example, a mouse model of diseases in which thetarget is naturally present in the mouse, for example bacterialinfection, viral infection, fungal infection, immune complex disease,self-antibody disease, hyperlipidemia, hyperglycemia, or for example, amouse model in which the target is administered to the mouse externally,e.g., by injection or by feeding. In these assays, the clearance of thetarget by the exogenous antigen-expressing EHC is assayed either byexamining the animal, e.g the plasma, the tissue, for reduction of thetarget, or by isolating or collecting the exogenous antigen-expressingEHC from the animal, e.g., from the blood, from the plasma, from atissue, and assaying the presence of the target on the exogenous antigenusing an in vitro assay including, but not limited to, for example, flowcytometry, immunohistochemistry, magnetic separation, radiography,colony-forming assays, microscopy.

In some embodiments, the exogenous antigen-expressing EHC is capable ofdelivering a suitable exogenous antigen to a specific subcellularcompartment, for example a lysosome.

For example, an exogenous antigen may be delivered to the lysosomalcompartment of a target cell, e.g., a macrophage. The successfuldelivery of the exogenous antigen to the lysosomal compartment of atarget cell is assessed by microscopy and the detection of punctatespots corresponding to a fluorescent exogenous antigen or fluorescentexogenous antigen detection agent. Alternatively or in addition, thesuccessful delivery of the exogenous antigen to the lysosomalcompartment of a target cell is assessed by microscopy and thecolocalization of a fluorescent exogenous antigen detection agent with afluorescent detection agent for a known lysosomal marker, e.g.,lysotracker, LAMP-1.

In some embodiments, the exogenous antigen is an enzyme that can degradetoxic components that have built up in the lysosome of a cell exhibitingthe genotype or phenotype of, or derived from a patient with, alysosomal storage disease. For example, the exogenous antigen is capableof degrading the toxic material built up in the cell and rescue the cellphenotype, e.g., preventing cell death.

The population of exogenous antigen-expressing EHCs can be assessed forthe inability of the EHCs to replicate, the ability of the EHCs tocirculate safely through the vasculature, and the lack of immunogenicityof the EHCs.

In some embodiments, the safety of the population of exogenousantigen-expressing EHCs is assessed by measuring the replicationpotential of the population of EHCs using a suitable in vitro or in vivoassay. For example, tests for a substantial inability of the exogenousantigen-expressing EHCs to self-replicate include: a) a susbstanitalinability to form a tumor when injected into an immunocompromised mouse;b) a substantial inability to form a colony when cultured in soft agar;c) a substantial inability to incorporate thymidine in a thymidineincorporation assay; d) a substantial lack of positive signal upontransfection with DNA encoding a fluorescent reporter, e.g., less than10%, 1%, 0.1%, 0.01%, 0.001%, 1 ppm, 100 ppb, 10 ppb, 1 ppb, 100 ppt, 10ppt, 1 ppt, or less than 1 ppt positive signal; e) a substantial lack ofpositive signal upon staining with a nuclear dye, e.g., less than 10%,1%, 0.1%, 0.01%; and 0.001%, 1 ppm, 100 ppb, 10 ppb, 1 ppb, 100 ppt, 10ppt, 1 ppt, or less than 1 ppt positive signal; f) a substantial lack ofpositive signal upon staining with cell markers of hematologicalmalignancy, e.g., CKIT, CD34, EpCam, e.g., less than 10%, 1%, 0.1%,0.01%, 0.001%, 1 ppm, 100 ppb, 10 ppb, 1 ppb, 100 ppt, 10 ppt, 1 ppt, orless than 1 ppt positive signal. In certain embodiments, exogenousantigen-expressing EHCs are provided that do not contain a substantialamount of a replicating nucleic acid.

In some embodiments, the safety of the population of exogenousantigen-expressing EHCs is assessed by measuring the ability of anadministered EHCto circulate in vivo (in the circulatory system of asubject) without causing substantial vascular occlusion or induction ofthe clotting cascade. Optionally, the circulation pharmacokinetics ofthe exogenous antigen-expressing EHCs may be assessed.

In one embodiment, the circulation pharmacokinetics of the exogenousantigen-expressing EHCs is assessed by injecting the EHC into an animalintravenously, such as a mouse. The mouse can be an NSG (nod-SCID-gamma)immunodeficient mouse. The mouse is depleted of macrophages prior toinjection with the EHC, e.g., by intraperitoneal injection of human redblood cells, or by intravenous injection with clodronate liposomes. Theexogenous antigen-expressing EHCs can be labeled with a fluoresecentdye, e.g., CFSE. After injection of the EHCs, blood is drawn and thenumber of exogenous antigen-expressing EHCs remaining is assessed by,e.g., flow cytometry, western blot, and the clearance rate of theexogenous antigen-expressing EHCs is deduced from these data. Human redblood cells can be injected into the same animal model as the exogenousantigen-expressing EHCs and the clearance rates of the EHCs and humanred blood cells are compared.

In one embodiment, the risk of activation of the clotting cascade by theexogenous antigen-expressing EHC is assessed with an in vitro assay. Inone embodiment, the exogenous antigen-expressing EHC is incubated withhuman blood and clotting cascade activation is assessed by measuring thetime required for coagulation in the presence of kaolin,negatively-charged phospholipids, and calcium (activated partialthromboplastn time (aPTT) test), see e.g., Exner and Rickard,Biomedicine 1977 27(2):62, or by measuring the time required forcoagulation in the presence of thromboplastin and calcium (prothrombintime (PT) test), see e.g., Jaques and Dunlop 1945, Am J Physiol 145:67.The normal range for the aPTT test is approximately 25-38 seconds. Thenormal range for the PT test is approximately 10-12 seconds.

In one embodiment, any adverse events induced by the exogenousantigen-expressing EHCs are assessed by injecting the EHCs into ananimal intravenously and assessing the activation of the clottingcascade. The level of clotting cascade induction is assessed by drawingblood and assessing the levels of clotting cascade components in theplasma by, e.g., Western Blot or ELISA. The clotting cascade componentsare typically fibrinogen breakdown products, e.g., fibrinopeptide A andfibrinopeptide B. For example, the level of clotting cascade inductionis assessed by drawing blood and assessing the levels of clottingactivity in the plasma by platelet activation assay, e.g., incubatingthe plasma with platelets and assessing the activation of the plateletsby flow cytometry, e.g., by staining for markers of activation, e.g., bystaining for PAC-1, CD62p, or CD40L.

In one embodiment, any adverse events induced by the exogenousantigen-expressing EHCs are assessed by injecting the EHCs into ananimal intravenously and assessing the activation of inflammatorypathways. The level of inflammation can be assessed by drawing blood andassessing the levels of inflammatory cytokines in the plasma by, e.g.,Western Blot or ELISA. In one embodiment, the inflammatory citokines areinterferon gamma, tumor necrosis factor alpha, or IL-12 fragment p70.

In one embodiment, any adverse events induced by the exogenousantigen-expressing EHCs are assessed by injecting the EHCs into ananimal intravenously and assessing the status of tissues, e.g., liver,spleen, heart, lungs, brain, skin, kidneys. The status of tissue can beassessed by gross necropsy, dissection of the tissue, histologicalstaining, and imaging by microscopy.

In one embodiment, the ability of the exogenous antigen-expressing EHCto circulate in vivo without causing substantial vascular occlusion oractivation of the clotting cascade is assessed by measuring thedeformability of the EHCs. The deformability of the exogenousantigen-expressing EHC is assessed using an in vitro assay. For example,the assay is an osmotic fragility assay. Mechanical fragility of theexogenous antigen-expressing EHC can be assessed by measuring thestructural integrity in response to shear stress in a Couett-typeshearing system. In one embodiment, the deformability of the exogenousantigen-expressing EHC is assessed using an Ektacytometer. In oneembodiment, the deformability of the exogenous antigen-expressing EHC isassessed by measuring the elongation index at a defined pressure bylaser diffraction using a laser-assisted optical rotational cellanalyzer (LORCA) instrument. In one embodiment, the deformability of theexogenous antigen-expressing EHC is assessed by measuring the transittime through a series of micron-scale constrictions of defineddimensions at a fixed pressure in a microfluidic device. In oneembodiment, the deformability of the exogenous antigen-expressing EHC isassessed by measuring the survival rate through a series of micron-scaleconstrictions of defined dimensions at a fixed pressure in amicrofluidic device. The microfluidic device can be selected from one ofthe following, including but not limited to, a poly dimethyl siloxane(PDMS) chip with micron-scale constrictions (e.g., Hoelzle et al. J VisExp 2014 91:e51474); a chip with funnel-shaped constrictions (e.g., Guoet al. Lab Chip 2012 12:1143); a PDMS chip with pillars (e.g., Zhang etal. PNAS 2012 109(46):18707); or an in vitro artificial spleen microbeadpacked column (Guillaume DePlaine et al., Blood 2011, 117(8)).

In one embodiment, the ability of the exogenous antigen-expressing EHCto circulate in vivo without causing substantial vascular occlusion oractivation of the clotting cascade is assessed by measuring the vascularocclusion of the EHCs. Vascular occlusion of the exogenousantigen-expressing EHC can be assessed using an in vitro assay. Forexample, the vascular occlusion of the exogenous antigen-expressing EHCis assessed using an ex vivo assay. The exogenous antigen-expressing EHCis incubated at a 1:1 ratio with reference human red blood cells andinduction of multi-cell rosettes are assessed by light microscopy incomparison to a reference assay with Rh-mismatched blood. The vascularocclusion of the exogenous antigen-expressing EHC is assessed bymeasuring the adhesion of the EHCs to human vascular endothelial cellsunder flow conditions, see e.g., Kaul D K, Finnegan E, and Bambino G A(2009) Microcirculation 16(1):97-111. Alternatively or in addition,vascular occlusion is assessed by measuring the peripheral resistanceunit (PRU) increase in an ex vivo perfusion assay of rat vascularendothelium, see e.g., Kaul, Fabry and Nagel, PNAS 1989, 86:3356.Further, vascular occlusion is assessed by intravital microscopy, seee.g., Zennadi et al. 2007 Blood 110(7):2708. Vascular occlusion may alsobe assessed by measuring flow rates and adhesion of the EHCs in vitrograduated height flow chambers, see e.g., Zennadi et al 2004, Blood104(12):3774.

In some embodiments, the safety of the population of exogenousantigen-expressing EHCs is assessed by measuring the immunogenicity ofthe population of EHCs using a suitable in vitro or in vivo assay.

For example, the population of exogenous antigen-expressing EHCs a) doesnot induce agglutination in a Coombs test using serum from the intendedrecipient subject or b) does not induce agglutination in a Coombs testusing pooled human serum.

In one embodiment, the population of exogenous antigen-expressing EHCsis derived from a progenitor cell that has been genotyped for thepredominant blood group antigens and matched to the blood group antigengenotype of the recipient.

In one embodiment, the population of exogenous antigen-expressing EHCscomprises an exogenous antigen or other exogenous protein that has lessthan 10%, 1%, 0.1%, 0.01%, 0.001%, or less than 0.001% predicted T cellreactivity by an in silico T cell epitope prediction algorithm.

In one embodiment, the population of exogenous antigen-expressing EHCscomprises an exogenous antigen or other exogenous protein that has lessthan 10%, 1%, 0.1%, 0.01%, 0.001%, or less than 0.001% reactivity in anin vitro T cell activation assay, e.g., Antitope Inc. EpiScreen assay.

For example, exogenous antigen-expressing EHCs derived from erythrocytescan be centrifuged and resuspended in appropriate solution (e.g.,standard AS-3 solution) for infusion into subjects in need thereof. Insome embodiments, the exogenous antigen-expressing EHCs to be infusedhave the same ABO type as that of the recipient to minimize the risk ofinfusion-associated immune reactions. The exogenous antigen-expressingEHCs can also be pretreated to remove blood type-specific antigens orotherwise reduce antigenicities. Methods suitable for this purposeinclude, but are not limited to, those described in U.S. PatentApplication Publication Nos. 20010006772 and 20030207247.

Therapeutic Compositions

Provided are compositions containing EHCs having effective levels ofexogenous antigen of interest. Such compositions contain a plurality ofEHCs, e.g., 1×10³ cells, or 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹,1×10¹⁰, 1×10¹¹, 1×10¹², or greater than 1×10¹² cells. EHCs of theinvention can, for example, be administered as packed red blood cells ina saline solution at a concentration of 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or greater than 90% mass to volume ratio (% m/v). Thetime of administration to a patient may range from 10 minutes to fourhours, or more.

The cultured EHCs of the invention can be stored in an appropriatebuffer, e.g. an FDA-approved anticoagulant preservative solution such asanticoagulant citrate-dextrose A (ACD-A), citrate-phosphate dextrose(CPD), Citratephosphate-dextrose-dextrose (CP2D), orcitrate-phosphate-dextrose-adenine (CPDA-1). The compositions may bestored for up to 21 days.

Alternatively, the cultured EHCs of the invention can be stored in anapproved additive solution, e.g. AS-1 (Adsol), AS-3 (Nutricel), AS-5(Optisol), or AS-7 (SOLX).

Alternatively, the cultured EHCs of the invention can stored in aglycerol cryoprotective solution. The compositions may be frozen andstored for up to 10 years. Frozen cells may be thawed and deglycerolizedby successive washing steps, for example with 0.9% sodium chloridebefore use.

Considering the life span of human and murine erythrocytes (120 and 50days, respectively (see, e.g. Khandelwal et al., Transfusion 2007), itmay be advantageous to stimulate artificial erythrocyte aging toincrease phagocytosis by antigen presenting cells. The most importantand physiological mechanism of erythrocyte removal from the circulationis immune-mediated (Singer et al., PNAS 1986) after exposure of newantigenic sites on RBC cell surface such as phosphatidylserineexternalization (Schroit et al., J Biol Chem 1985) or Band 3 proteinclustering (Kay, PNAS 1975; Turrini et al., J Biol Chem 1991) inducedartificially with calcium ionophore or BS3 chemical treatments,respectively.

The cultured EHCs of the invention may be treated with aphagocytosis-inducing agent such as, e.g. calcium ionophore or BS3. Theplurality of cultured EHCs of the invention may comprise EHCs that havebeen treated with a phagocytosis-inducing agent, such as, e.g. calciumionophore or BS3, for differing lengths of time, such that uponadministration to a subject, the plurality of cultured EHCs of theinvention are phagocytosed at different rates, e.g. continuously overthe course of one day or several days rather than as a bolus.

Provided herein are compositions and pharmaceutical compositionscomprising a plurality of cultured EHCs that comprise an exogenousantigen of interest. The compositions and pharmaceutical compositionsmay comprise a solution of appropriate storage buffer such as, e.g.anticoagulant citrate-dextrose A. The compositions and pharmaceuticalcompositions comprising the plurality of cultured EHCs that comprise anexogenous antigen of interest may additionally comprise an approvedadditive such as, e.g. Adsol. The compositions and pharmaceuticalcompositions comprising the plurality of cultured EHCs that comprise anexogenous antigen of interest may additionally comprise a glycerolcryoprotective solution for frozen storage.

Provided herein are EHCs comprising an exogenous antigen of interestselected from one or more of the antigens of Table F, Table G, Table H,Table I and Table J, such as e.g. myelin basic protein, proteolipidprotein, myelin oligodendrocyte glycoprotein, pancreatic beta cellantigen, insulin, flagellin, gluten, 2S albumin, hyalauronidase, factorVIII, factor IX, and anti-TNFa, adenosine deaminase, L-asparaginase,rasburicase, antithymocyte globulin, L-arginase, L-methionase,preproinsulin, proinsulin, insulin, GAD65, GAD67, IA-2, IA-2β,thyroglobulin, thyroid peroxidase, thyrotropin receptor, myelinoligodendrocyte glycoprotein, proteolipid protein, collagen II, collagenIV, acetylcholine receptor, matrix metalloprotein 1 and 3, molecularchaperone heat-shock protein 47, fibrillin-1, PDGF receptor a, PDGFreceptor β, nuclear protein SS-A, conarachin (Ara h 1), allergen II (Arah 2), arachis agglutinin (Ara h 6), a-lactalbumin (ALA),lactotransferrin, glutein, low molecular weight glutein, a-gliadin,γ-gliadin, hordein, secalin, avenin and combinations thereof. Aplurality of EHCs comprising an exogenous antigen of interest may beprovided as a composition or pharmaceutical composition.

Provided herein are expression vectors encoding one or more antigens ofTable F, Table G, Table H, Table I and Table J, such as e.g. myelinbasic protein, proteolipid protein, myelin oligodendrocyte glycoprotein,pancreatic beta cell antigen, insulin, flagellin, gluten, Ara h2, 2Salbumin, hyalauronidase, factor VIII, factor IX, and anti-TNFa,optionally fused to one of the endogenous erythroid proteins of Table C.

Provided herein are pharmaceutical compositions comprising exogenousantigen-expressing EHCs that are suitable for administration to asubject. The pharmaceutical compositions generally comprise a populationof exogenous antigen-expressing EHCs and a pharmaceutically-acceptablecarrier in a form suitable for administration to a subject.Pharmaceutically-acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionscomprising a population of exogenous antigen-expressing EHCs. (See,e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa. 18th ed. (1990)). The pharmaceutical compositions are generallyformulated as sterile, substantially isotonic and in full compliancewith all Good Manufacturing Practice (GMP) regulations of the U.S. Foodand Drug Administration.

Pharmaceutically-acceptable excipients include excipients that aregenerally safe, non-toxic, and desirable, including excipients that areacceptable for veterinary use as well as for human pharmaceutical use.Such excipients can be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

Examples of carriers or diluents include, but are not limited to, water,saline, Ringer's solutions, dextrose solution, and 5% human serumalbumin. The use of such media and compounds for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or compound is incompatible with the exogenous antigen-expressingEHCs described herein, use thereof in the compositions is contemplated.Supplementary therapeutic agents may also be incorporated into thecompositions. Typically, a pharmaceutical composition is formulated tobe compatible with its intended route of administration. The exogenousantigen-expressing EHCs can be administered by parenteral, topical,intravenous, oral, subcutaneous, intraarterial, intradermal,transdermal, rectal, intracranial, intraperitoneal, intranasal;intramuscular route or as inhalants. The exogenous antigen-expressingEHCs can optionally be administered in combination with othertherapeutic agents that are at least partly effective in treating thedisease, disorder or condition for which the exogenousantigen-expressing EHCs are intended.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial compounds such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating compounds such as ethylenediaminetetraacetic acid (EDTA);buffers such as acetates, citrates or phosphates, and compounds for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, e.g., water,ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, e.g., by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalcompounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic compounds, e.g., sugars, polyalcohols such as manitol,sorbitol, sodium chloride in the composition. Prolonged absorption ofthe injectable compositions can be brought about by including in thecomposition a compound which delays absorption, e.g., aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating theexogenous antigen-expressing EHCs in an effective amount and in anappropriate solvent with one or a combination of ingredients enumeratedherein, as desired. Generally, dispersions are prepared by incorporatingthe exogenous antigen-expressing EHCs into a sterile vehicle thatcontains a basic dispersion medium and any desired other ingredients. Inthe case of sterile powders for the preparation of sterile injectablesolutions, methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The exogenous antigen-expressing EHCs can be administered in the form ofa depot injection or implant preparation which can be formulated in sucha manner to permit a sustained or pulsatile release of the exogenousantigen-expressing EHCs, their exogenous antigen(s) and/or theiroprional payload(s).

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, theexogenous antigen-expressing EHCs can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash,wherein the compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingcompounds, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegratingcompound such as alginic acid, Primogel, or corn starch; a lubricantsuch as magnesium stearate or Sterotes; a glidant such as colloidalsilicon dioxide; a sweetening compound such as sucrose or saccharin; ora flavoring compound such as peppermint, methyl salicylate, or orangeflavoring.

For administration by inhalation, the exogenous antigen-expressing EHCsare delivered in the form of an aerosol spray from pressured containeror dispenser which contains a suitable propellant, e.g., a gas such ascarbon dioxide, or a nebulizer.

Systemic administration of compositions comprising exogenousantigen-expressing EHCs can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, e.g., fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the modified red blood cells are formulated into ointments, salves,gels, or creams as generally known in the art.

The exogenous antigen-expressing EHCs can also be prepared aspharmaceutical compositions in the form of suppositories (e.g., withconventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In some embodiments, the exogenous antigen-expressing EHCs are preparedwith carriers that will decrease the rate with which exogenousantigen-expressing EHCs are eliminated from the body of a subject. Forexample, controlled release formulation are suitable, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc.

In one embodiment the pharmaceutical composition comprising exogenousantigen-expressing EHCs is administered intravenously into a subjectthat would benefit from the pharmaceutical composition. In otherembodiments, the composition is administered to the lymphatic system,e.g., by intralymphatic injection or by intranodal injection (see e.g.,Senti et al., 2008 PNAS 105(46):17908), or by intramuscular injection,by subcutaneous administration, by direct injection into the thymus, orinto the liver.

In one embodiment, the pharmaceutical composition comprising exogenousantigen-expressing EHCs is administered as a liquid suspension. In oneembodiment the pharmaceutical composition is administered as acoagulated formulation that is capable of forming a depot followingadministration, and in a preferred embodiment slowly release exogenousantigen-expressing EHCs into circulation, or in a preferred embodimentremain in depot form.

In one embodiment, the pharmaceutical composition comprising exogenousantigen-expressing EHCs is stored using methods and buffer compositionsthat are capable of maintaining viability of the exogenousantigen-expressing EHCs. For example, deoxygenation prior to storage tomaintain an anaerobic state, manipulation of pH, supplementation ofmetabolic precursors, manipulation of osmotic balance, increasing of thevolume of the suspending medium, and/or reduction of oxidative stress byadding protective molecules can be used to maintain the viability of theexogenous antigen-expressing EHCs. Several studies employing acombination of these strategies have reported maintenance of viabilityof erythrocytes allowing an extension of storage beyond 6 weeks (seee.g., Yoshida and Shevkoplyas, Blood Transfus 2010 8:220).

Pharmaceutically acceptable carriers or excipients may be used todeliver the exogenous antigen-expressing EHCs described herein.Excipient refers to an inert substance used as a diluent or vehicle.Pharmaceutically acceptable carriers are used, in general, with acompound so as to make the compound useful for a therapy or as aproduct. In general, for any substance, a pharmaceutically acceptablecarrier is a material that is combined with the substance for deliveryto a subject. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable. Insome cases the carrier is essential for delivery, e.g., to solubilize aninsoluble compound for liquid delivery; a buffer for control of the pHof the substance to preserve its activity; or a diluent to prevent lossof the substance in the storage vessel. In other cases, however, thecarrier is for convenience, e.g., a liquid for more convenientadministration. Pharmaceutically acceptable salts of the compoundsdescribed herein may be synthesized according to methods known to thoseskilled in the arts.

Typically, pharmaceutically acceptable compositions are highly purifiedto be free of contaminants, are biocompatible and not toxic, and aresuited to administration to a subject. If water is a constituent of thecarrier, the water is highly purified and processed to be free ofcontaminants, e.g., endotoxins.

The pharmaceutically acceptable carrier may be lactose, dextrose,sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate,alginates, gelatin, calcium silicate, micro-crystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose,methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesiumstearate, and/or mineral oil, but is not limited thereto. Thepharmaceutical composition may further include a lubricant, a wettingagent, a sweetener, a flavor enhancer, an emulsifying agent, asuspension agent, and/or a preservative.

Provided are pharmaceutical compositions containing exogenousantigen-expressing EHCs having effective levels of exogenous antigens.Such compositions contain a plurality of exogenous antigen-expressingEHCs, e.g., 1×10³ EHCs, or 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹,1×10¹⁰, 1×10¹¹, 1×10¹², or greater than 1×10¹² EHCs. In specificexamples, exogenous antigen-expressing EHCs generated from EHCs may beadministered as packed red blood cells in a saline solution at aconcentration of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greaterthan 90% mass to volume ratio (%m/v). The time of administration to apatient may range from 10 minutes to four hours, or more.

In specific examples, exogenous antigen-expressing EHCs generated fromEHCs can be stored in an appropriate buffer, e.g., an FDA-approvedanticoagulant preservative solution such as anticoagulantcitrate-dextrose A (ACD-A), citrate-phosphate dextrose (CPD),Citratephosphate-dextrose-dextrose (CP2D), orcitrate-phosphate-dextrose-adenine (CPDA-1). The compositions may bestored for up to 21 days.

Alternatively, exogenous antigen-expressing EHCs generated from EHCs canbe stored in an approved additive solution, e.g., AS-1 (Adsol), AS-3(Nutricel), AS-5 (Optisol), or AS-7 (SOLX).

Alternatively, exogenous antigen-expressing EHCs generated from EHCs canstored in a glycerol cryoprotective solution. The compositions may befrozen and stored for up to 10 years. Frozen cells may be thawed anddeglycerolized by successive washing steps, for example with 0.9% sodiumchloride before use.

Provided herein are compositions and pharmaceutical compositionscomprising a plurality of cultured EHCs that comprise an exogenousantigen. The compositions and pharmaceutical compositions may comprise asolution of appropriate storage buffer such as, e.g., anticoagulantcitrate-dextrose A. The compositions and pharmaceutical compositionscomprising the plurality of cultured EHCs that comprise an exogenousantigen may additionally comprise an approved additive such as, e.g.,Adsol. The compositions and pharmaceutical compositions comprising theplurality of cultured EHCs that comprise exogenous antigen mayadditionally comprise a glycerol cryoprotective solution for frozenstorage.

In one embodiment, the exogenous antigen-expressing EHC is able to forma multi-complex aggregate, e.g., a dimer, a trimer, a multimer, withanother exogenous antigen-expressing EHC.

In one embodiment the exogenous antigen-expressing EHC is able to form amulti-complex aggregate, e.g., a dimer, a trimer, a multimer, withcomponent of the circulatory system, e.g an erythrocyte, a reticulocyte,a platelet, a macrophage, a lymphocyte, a T cell, a B cell, a mast cell.

The dosage and frequency of the administration of the exogenousantigen-expressing EHCs and pharmaceutical compositions thereof can bedetermined by the attending physician based on various factors such asthe severity of disease, the patient's age, sex and diet, the severityof any inflammation, time of administration, and other clinical factors.In one example, an intravenous administration is initiated at a dosewhich is minimally effective, and the dose is increased over apre-selected time course until a positive effect is observed.Subsequently, incremental increases in dosage are made limiting tolevels that produce a corresponding increase in effect while taking intoaccount any adverse affects that may appear.

Non-limited examples of suitable dosages can range, for example, from1×10³ to 1×10¹⁴, from 1×10³ to 1×10⁷, from 1×10⁵ to 1×10⁶, from 1×10⁷ to1×10¹¹, from 1×10⁸ to 1×10⁹, from 1×10¹⁰ to 1×10¹⁴, from 1×10¹¹ to1×10¹³, or from 5×10¹¹ to 5×10¹² exogenous antigen-expressing EHCs.Specific examples include about 1×10³, 2×10³, 3×10³, 4×10³, 5×10³,6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴,7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵,8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶,9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷,1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹,2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰,3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹,3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², or moreexogenous antigen-expressing EHCs. Each dose of exogenousantigen-expressing EHCs can be administered at intervals such as oncedaily, once weekly, twice weekly, once monthly, or twice monthly. EachEHC may express a range of antigen molecules, for example, from about100 to 10{circumflex over ( )}7, or from about 10{circumflex over ( )}3to 10{circumflex over ( )}6. Specific examples include about 1000, 3000,5000, 1×10{circumflex over ( )}4, 3×10{circumflex over ( )}4,5×10{circumflex over ( )}4, 1×10{circumflex over ( )}5, 3×10{circumflexover ( )}5, 5×10{circumflex over ( )}5, 1×10{circumflex over ( )}6,3×10{circumflex over ( )}6, 5×10{circumflex over ( )}6, 1×10{circumflexover ( )}7, or more exogenous antigen molecules per EHC.

“EHC-based proportional dosage” is the number of exogenousantigen-expressing EHCs administered as a dose relative to a naturallyoccurring quantity of circulating entities. The circulating entities maybe cells, e.g., erythrocytes, reticulocytes, or lymphocytes, or targets,e.g., antigens, antibodies, viruses, toxins, cytokines, etc. The unitsare defined as exogenous antigen-expressing EHC per circulating entity,ie SCMRC/CE. This dosage unit may include 10⁻⁷, 10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³,10⁻², 10⁻¹, 1, 10, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹.

The pharmaceutical compositions described herein comprise an exogenousantigen-expressing EHC and optionally a pharmaceutically active ortherapeutic agent. The therapeutic agent can be a biological agent, asmall molecule agent, or a nucleic acid agent.

Dosage forms are provided that comprise a pharmaceutical compositioncomprising an exogenous antigen-expressing EHC described herein. In someembodiments, the dosage form is formulated as a liquid suspension forintravenous injection.

Medical devices are provided that comprise a container holding apharmaceutical composition comprising an exogenous antigen-expressingEHC described herein and an applicator for intravenous injection of thepharmaceutical composition to a subject.

Medical kits are provided that comprise a pharmaceutical compositioncomprising an exogenous antigen-expressing EHC described herein and amedical device for intravenous injection of the pharmaceuticalcomposition to a subject.

A pharmaceutically acceptable suspension of exogenous antigen-expressingEHCs is preferably packaged in a volume of approximately 10 toapproximately 250 ml. The packaging can be a syringe or an IV bagsuitable for transfusions. Administration of the suspension is carriedout, e.g., by intravenous or intra-arterial injection, optionally usinga drip from an IV bag or the like. The administration is typicallycarried out intravenously in the arm or via a central catheter. Foradministrations exceeding 50 ml use of a drip is preferred.

Treatment of Diseases

Provided are methods of inducing immune tolerance. The methods includeadministering to a subject in need of induction of immune tolerance apharmaceutical composition of the erythrocyte cells that comprise anexogenous antigen of interest provided herein in an amount and/or adosing frequency sufficient to induce immune tolerance in the subject.

The pharmaceutical compositions of the invention provide erythrocytecells that comprise an exogenous antigen of interest that are useful topromote or enhance immune tolerance Immune tolerance may be used totreat, prevent, or reduce the severity of a disease, disorder, orcondition associated with immune activation.

Diseases of immune activation include autoimmune diseases, such as, e.g.multiple sclerosis, type 1 diabetes, rheumatoid arthritis, andmembranous nephritis, and those listed in Table F. Diseases of immuneactivation also include inflammatory diseases, such as, e.g. Crohn'sdisease, ulcerative colitis, celiac disease, or other idiopathicinflammatory bowl diseases, and those listed in Table G. Diseases ofimmune activation also include allergic diseases, such as, e.g. asthma,peanut allergy, shellfish allergy, pollen allergy, milk protein allergy,insect sting allergy, and latex allergy, and those listed in Table H.Diseases of immune activation also include immune activation in responseto a therapeutic protein, administered to treat a primary condition,that lessens the efficacy of the therapeutic protein, such as, e.g.,clotting factor VIII in hemophilia A, clotting factor IX in hemophiliaB, anti-tumor necrosis factor alpha (TNFa) antibodies in rheumatoidarthritis and other inflammatory diseases, glucocerebrosidase inGaucher's disease, or asparaginase in acute lymphoblastic leukemia(ALL), and those listed in Table I, Table J, Table 5, and Table 7.

Further provided are methods for treating an immune activation disease.The methods include administering to a subject in need of induction oftreatment a pharmaceutical composition of the erythrocyte cells thatcomprise an exogenous antigen of interest provided herein in an amountsufficient to treat the immune activation disease. For example, asubject that has or is suspected of having an immune activation diseasesuch as autoimmune disease, inflammatory disease or allergic diseasewould benefit from the treatment methods provided.

In some embodiments a patient is suffering from an autoimmune disease orcondition or a self-antibody mediated disease or condition, in which thepatient's immune system is active against an endogenous molecule, forexample a protein antigen, such that the immune system attacks theendogenous molecule, induces inflammation, damages tissue, and otherwisecauses the symptoms of the autoimmune or self-antibody disease orconditions. The immune response might be driven by antibodies that bindto the endogenous molecule, or it may be driven by overactive T cellsthat attack cells expressing the endogenous molecule, or it may bedriven by other immune cells such as regulatory T cells, NK cells, NKTcells, or B cells. In these embodiments, the antigenic protein or afragment thereof may be expressed on an enucleated hematopoietic cell ofthe invention. A population of these cells, when administered once ormore to the patient suffering from the disease or condition, would besufficient to induce tolerance to the antigenic protein such that it nolonger induced activation of the immune system, and thus would treat orameliorate the symptoms of the underlying disease or condition.

For example, a patient suffering from acquired thromboticthrombocytopenic purpura (TTP) has an aberrant self-antibody mediateddisease in which antibodies are generated against endogenous ADAMTS13protein rendering it ineffective at performing its von WillebrandFactor-cleaving activities, which results in microthrombi formingthroughout the vasculature and consequent thrombocytopenia. In thisembodiment, the ADAMTS13 antigen is expressed on an eucleatedhematopoietic cell and administered to a patient suffering from TTP inan amount effective to induce tolerance to ADAMTS13, thus reducing thequantity of inhibitory anti-ADAMTS13 self-antibodies in circulation andrestoring the ability of the body to cleave von Willebrand Factor thusreducing the symptoms of the disease. In a preferred embodiment, onlythe antigenic fragment of ADAMTS13 is expressed on the enucleatedhematopoietic cell. In a preferred embodiment, the full-length ADAMTS13is expressed on the enucleated hematopoietic cell. In a preferredembodiment, the full-length ADAMTS13 is expressed on the enucleatedhematopoietic cell and is enzymatically active, such that theadministered cell product is able to both induce tolerance and alsotherapeutically cleave von Willebrand Factor.

In another example, a patient suffering from atypical hemolytic anemicsyndrome (aHUS) has an aberrant self-antibody response to the endogenousprotein Complement Factor H (CFH), preventing CFH from performing it'scomplement regulatory function. As a result, complement overactivationoccurs in the vasculature leading to intravascular hemolysis. In thisembodiment, the CFH antigen is expressed on an enucleated hematopoieticcell and administered to a patient suffering from aHUS in an amounteffective to induce tolerance to CFH, thus reducing the quantity ofinhibitory anti-CFH self-antibodies in circulation and restoring theability of the body to inhibit complement thus reducing the symptoms ofthe disease. In a preferred embodiment, only the antigenic fragment ofCFH is expressed on the enucleated hematopoietic cell. In a preferredembodiment, the full-length CFH is expressed on the enucleatedhematopoietic cell. In a preferred embodiment, the full-length CFH isexpressed on the enucleated hematopoietic cell and is therapeuticallyactive, such that the administered cell product is able to both inducetolerance and also therapeutically promote complement regulation.

In another example, a patient suffering from multiple sclerosis (MS) hasan autoimmune response to the polypeptide myelin that sheathes neurons.As a result, T cells attack the myelin and the resultant inflammationcauses demyelination of the nerve fibers and impairs the ability forelectrical signals to be sent along the nerves leading to the symptomsof multiple sclerosis. In this embodiment, the myelin antigen isexpressed on an enucleated hematopoietic cell and administered to apatient suffering from MS in an amount effective to induce tolerance tomyelin antigen, thus reducing the anti-myelin immune response andrestoring the ability of the body to send electrical impulses downmyelinated nerve fibers, thus reducing the symptoms of the disease. In apreferred embodiment, only one or more antigenic fragments of myelin areexpressed on the enucleated hematopoietic cell. In a preferredembodiment, the full-length myelin protein is expressed on theenucleated hematopoietic cell.

In another example, a patient suffering from type 1 diabetes (T1D) hasan autoimmune response to the beta islet cells of the pancreas. As aresult, T cells kill the beta islet cells reducing or eliminating thepancreas' ability to produce and secrete insulin, which leads to thesymptoms and pathology of T1D. In this embodiment, the beta cell antigenis expressed on an enucleated hematopoietic cell and administered to apatient suffering from T1D in an amount effective to induce tolerance tobeta cell antigen, thus reducing the anti-beta cell immune response andrestoring the ability of the pancreas to produce and secrete insulin,thus reducing the symptoms of the disease. In a preferred embodiment,only one or more antigenic fragments of beta cell antigen are expressedon the enucleated hematopoietic cell. In a preferred embodiment, thefull-length beta cell protein is expressed on the enucleatedhematopoietic cell.

Further provided are methods of reducing or alleviating an immuneactivation in response to a therapeutic protein treatment regimen. Themethods include administering to a subject in need of reducing oralleviating an immune activation in response to a therapeutic proteintreatment regimen a pharmaceutical composition of the erythrocyte cellsthat comprise an exogenous antigen of interest provided herein in anamount sufficient to reduce or alleviate the immune activation inresponse to a therapeutic protein treatment regimen.

In some embodiments a patient is suffering from a disease or conditionfor which a therapeutic protein can be administered to treat orameliorate the symptoms of the disease or condition, but the therapeuticprotein is immunogenic such that the patient elicits an immune responseagainst the therapeutic protein such that it is no longer effective attreating or ameliorating the original disease. For example, theimmunogenic therapeutic protein might be derived from a non-humansource, e.g. bovine, porcine, or non-human primate, or from anon-mammalian source, e.g. bacterial, fungal, or plat-derived, or theimmunogenic therapeutic protein may be derived from a human source butthe repetitive exposure and dosing might be sufficient to induceimmunogenicity. The immune response might be driven by antibodies thatbind to the immunogenic therapeutic protein and inhibit its function(neutralizing antibodies) or that bind to the immunogenic therapeuticprotein and trigger its clearance by other immune cells (opsonizingantibodies). In these embodiments, the immunogenic therapeutic proteinor an antigenic fragment thereof may be expressed on an enucleatedhematopoietic cell of the invention. A population of these cells, whenadministered once or more to the patient suffering from the disease,would be sufficient to induce tolerance to the immunogenic therapeuticprotein such that it was no longer neutralized or opsonized by theimmune system. In one preferred embodiment, the immunogenic therapeuticprotein expressed on the surface of the enucleated hematopoietic cell ofthe invention is therapeutically active on the cell in circulation, suchthat the composition of cell and protein is able to both inducetolerance and treat or ameliorate the symptoms of the underlying diseaseor condition when administered to the patient. In another preferredembodiment the antigenic fragment of the immunogenic therapeutic proteinexpressed on the surface of the enucleated hematopoietic cell of theinvention is not therapeutically active on the cell in circulation, suchthat the composition of cell and protein is able to induce tolerancewhen administered to the patient but a separate formulation ofimmunogenic therapeutic protein is administered to treat or amelioratethe symptoms of the underlying disease or condition.

For example, a patient suffering from hemophilia A requires infusions ofclotting factor VIII (FVIII) to restore proper coagulation. However manypatients develop neutralizing antibodies to FVIII despite it being humanderived, which render the therapeutic ineffective and lead to alife-threatening risk of bleeding. In one embodiment, an exogenous FVIIIexpressing enucleated hematopoietic cell is administered to the patientsuffering from hemophilia A such that (1) the levels of circulatingactive FVIII are restored to a level necessary to ameliorate thesymptoms and prevent severe uncontrolled bleeding, and such that (2)tolerance is induced to FVIII.

In another example, a patient suffering from rheumatoid arthritisrequires injections of anti-TNFa antibody to reduce the inflammationassociated with that disease. However may patients develop neutralizingantibodies against the anti-TNFa antibody that render the therapeuticantibody ineffective. In this instance, the patient typically suffers aworsening of symptoms and either has to increase the dose of theanti-TNFa antibody or switch to a different anti-TNFa antibody with adifferent coding sequence of amino acids. In this embodiment, anenucleated hematopoietic cell expressing an antigenic fragment ofanti-TNFa is administered to a patient with rheumatoid arthritis who hasdeveloped neutralizing antibodies against the anti-TNFa antibody. Thecomposition is administered at a dose sufficient to induce tolerance tothe anti-TNFa antibody, allowing effective administration of anti-TNFato reduce the circulating TNFa levels and thus reduce the symptoms ofrheumatoid arthritis in the patient.

In another example, a patient suffering from phenylketonuria (PKU) istreated with pegylated phenylalanine ammonia lyase (PAL), a non-humanenzyme. The patient develops opsonizing and neutralizing antibodiesagainst PAL that also elicit an allergic reaction upon administration ofthe therapeutic protein. This immune response not only renders the PALineffective, it also threatens the health of the patient. In oneembodiment, enucleated hematopoietic cells expressing exogenous PAL areadministered to a patient suffering from PKU in an amount sufficient toinduce tolerance to PAL. In a preferred embodiment, the cell-expressedPAL is active on the cell, and the composition is able to reduce thecirculating levels of phenylalanine and treat or ameliorate the symptomsof phenylketonuria in addition to preventing a dangerous immune reactionagainst PAL. In another preferred embodiment, an antigenic fragment ofPAL is expressed on the enucleated hematopoietic cell, and thiscell-expressed fragment is not therapeutically active, so a separateformulation of PAL is administered to the patient to treat or amelioratethe symptoms of phenylketonuria. The tolerance-inducing cell compositioncan be administered prior to administering the therapeutic formulationof PAL, or the tolerance-inducing cell composition can be administeredconcurrent to the administration of the therapeutic formulation of PAL.

In some embodiments a patient is suffering from an allergic disease, forexample an allergy to animal dander, black walnut, brazil nut, cashewnut, chestnut, dust mites, egg, english walnut, fish , hazelnut, insectvenom, latex, milk, mold, peanuts, pollen, grass, shellfish, soy, treenuts, or wheat. A patient suffering from an allergy may mount an immuneresponse upon contact with the antigenic fragment of the allergen, forexample through diet, skin contact, injection, or environmentalexposure. The immune response may involve IgE antibody, sensitized mastcells, degranulation, histamine release, and anaphylaxis, as well ascanonical immune cells like T cells, B cells, dendritic cells, Tregulatory cells, NK cells, neutrophils, and NKT cells. The allergicreaction may cause discomfort or it may be severe enough to cause death,and thus requires constant vigilance on the part of the sufferer as wellas his or her family and caretakers. In these embodiments, the antigenicprotein or a fragment thereof may be expressed on an enucleatedhematopoietic cell of the invention. A population of these cells, whenadministered once or more to the patient suffering from the allergicdisease or condition, would be sufficient to induce tolerance to theantigenic protein such that it no longer induced activation of theimmune system upon exposure, and thus would treat or ameliorate thesymptoms of the underlying allergic disease or condition.

In one example a patient suffering from peanut allergy has an immuneresponse following exposure to peanut antigen AraH1. In this embodiment,AraH1 is expressed on an enucleated hematopoietic cell and administeredto a patient suffering from peanut allergy in an amount effective toinduce tolerance to AraH1 antigen, thus reducing the allergic immuneresponse and restoring the ability of the individual to safely consumepeanuts, thus reducing the symptoms of the disease. In a preferredembodiment, only one or more antigenic fragments of AraH1 are expressedon the enucleated hematopoietic cell. In a preferred embodiment, thefull-length AraH1 protein is expressed on the enucleated hematopoieticcell.

Certain aspects of the invention relate to EHCs that comprise antigenthat is recognized by immune cells in human leukocyte antigen (HLA)mismatch-mediated diseases, such as, e.g. graft-versus-host disease ororgan transplant rejection.

In some embodiments, a patient is suffering from a disease or conditionof human leukocyte antigen (HLA)-mismatch in which immune cells areactivated against HLA antigens on a tissue and attack the tissue. Thiscommonly occurs following allogeneic transplantation of an organ ortissue from a donor who is not a perfect match and leads to the medicalcondition of transplant rejection, in which the patient's immune systemattacks the foreign tissue or organ and causes the transplanted organ ortissue to die. Another common HLA-mismatch condition isGraft-versus-Host Disease (GVHD) in which a patient has received anallogeneic bone marrow transplantation from a donor who is not a perfectmatch and in which the transplanted immune cells (graft) becomeactivated and attack the recipients organs (host), which are recognizedas foreign, causing the damage of host tissues and organs and leading tosevere consequences including death. HLA-mismatch immune activation istypically mediated by T cells, but can also involve T regulatory cells,NK cells, NKT cells, B cells, antibodies, dendritic cells, monocytes,macrophages, and neutrophils. In these embodiments, the antigenic HLAmolecule or a fragment thereof may be expressed on an enucleatedhematopoietic cell of the invention. A population of these cells, whenadministered once or more to the patient suffering from the HLA-mismatchdisease or condition, would be sufficient to induce tolerance to theantigenic HLA molecule such that it no longer induced activation of theimmune system and thus would treat or ameliorate the symptoms of theunderlying HLA-mismatch disease or condition, for example the survivalof the transplanted organ or the survival of the patient. In a preferredembodiment, the HLA molecule expressed on the surface of the cell alsocontains a peptide loaded into the HLA molecule.

The erythrocyte cells that comprise an exogenous antigen that are usedfor the methods described herein can be derived autologously, i.e. fromthe same subject, or may be allogeneically derived, i.e. from adifferent cell donor.

The pharmaceutical compositions may be administered to the subject forexample by intravenous transfusion or intramuscular injection.

Other Embodiments

It is to be understood that this invention is not limited to particularmethods, reagents, compounds, compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only,and is not intended to be limiting.

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

In some embodiments, the exogenous antigen-expressing EHC comprising aCR1 exogenous antigen is not generated in a mouse and/or is notgenerated from mouse erythroid cells. In some embodiments, the exogenousantigen-expressing EHC comprising a CR1 exogenous antigen is notgenerated in a laboratory animal and/or is not generated from anerythroid cells derived from a laboratory animal. In some embodiments,the exogenous antigen-expressing EHC is generated from megakaryocytes orplatelets. In some embodiments, the exogenous antigen-expressing EHC isgenerated from an erythroid cell, such as, e.g. an erythrocyte or areticulocyte. In some embodiments, the exogenous antigen-expressing EHCis not generated from a neutrophil, an eosinophil, or a basophil. Insome embodiments, the exogenous antigen-expressing EHC is not generatedfrom a monocyte or a macrophage. In some embodiments, the exogenousantigen-expressing EHC is not generated from a CD34⁺Thy-1⁺ hematopoieticstem cell or cell populations enriched in CD34⁺Lin⁻ or CD34⁺Thy-1⁺Lin⁻cells. In some embodiments, the exogenous antigen-expressing EHC doesnot comprise an exogenous antigen comprising an extracellular domain ofan HIV coreceptor. In some embodiments, the exogenous antigen-expressingEHC does not comprise an exogenous antigen capable of binding to avirus. In some embodiments, the exogenous antigen-expressing EHC doesnot comprise an exogenous antigen comprising CD4. In some embodiments,the exogenous antigen-expressing EHC does not comprise an exogenousantigen comprising an HIV coreceptor. In some embodiments, the exogenousantigen-expressing EHC does not comprise an exogenous antigen comprisingCXCR4, CCR5, CCR1, CCR2, CCR3, CCR4, CCR8, CXCR1, CXCR2, CXCR3, CXCR6,GPR15, APJ, CMKLR1, or CX3CR1 or any combination thereof. In someembodiments, the exogenous antigen-expressing EHC does not contain anexogenous nucleic acid encoding an adenosine deaminase antigen. In someembodiments, the exogenous antigen-expressing EHC does not comprise anexogenous antigen comprising adenosine deaminase (ADA). In someembodiments, the exogenous antigen-expressing EHC does not comprise anexogenous nucleic acid encoding an oncogene. In some embodiments, theexogenous antigen-expressing EHC does not comprise an exogenous antigencomprising oncogene. In some embodiments, the exogenousantigen-expressing EHC does not contain an exogenous nucleic acidencoding cdx1, cdx2, or cdx4. In some embodiments, the exogenousantigen-expressing EHC does not comprise an exogenous antigen comprisingcdx1, cdx2, or cdx4, or any combination thereof. In some embodiments,the exogenous antigen-expressing EHC does not comprise an exogenousantigen comprising a chimeric polypeptide comprising a ligand bindingdomain. In some embodiments, the exogenous antigen-expressing EHC doesnot comprise an exogenous antigen comprising an S domain that is capableof binding a ligand. In some embodiments, the exogenousantigen-expressing EHC does not comprise an exogenous antigen comprisingCD3ζ, CD3η, an IL-2 receptor, an IL-3 receptor, an IL-4 receptor, anIL-7 receptor, an IL-11 receptor, an IL-13 receptor, a GM-CSF receptor,a LIF receptor, a CNTF receptor, an oncostatin M receptor, a TGF-βreceptor, an EGF receptor, ATR2/neu, a HER2/neu, a HER3/c-erbB-3, Xmrk,an insulin receptor, an IGF-1 receptor, IRR, PDGF receptor, a CSF-1receptor, c-kit, STK-1/flk-2, an FGF receptor, flg, bek, an NGFreceptor, Ror1 and Ror2 or any combination thereof. In some embodiments,the exogenous antigen-expressing EHC does not comprise an exogenousantigen comprising E6 or E7 genes of human papillomavirus. In someembodiments, the exogenous antigen-expressing EHC does not comprise anexogenous antigen comprising a tumor antigen. In some embodiments, theexogenous antigen-expressing EHC does not comprise an exogenous antigencomprising glucocerebrosidase. In some embodiments, the exogenousantigen-expressing EHC does not comprise an exogenous antigen comprisingasparaginase. In some embodiments, the exogenous antigen-expressing EHCdoes not comprise an exogenous antigen comprising arginine deiminase. Insome embodiments, the exogenous antigen-expressing EHC does not comprisea fusion molecule capable of promoting fusion of the exogenousantigen-expressing EHC to a target cell that is i) different from and/orii) acts independent of the exogenous antigen, wherein the exogenousantigen is capable of interacting with a target. In some embodiments,the exogenous antigen-expressing EHC does not comprise an exogenousantigen comprising Syncytin-1. In some embodiments, the exogenousantigen-expressing EHC does not comprise a photosensitive syntheticcompound, such as, e.g. a compound that can be activated by photons orquenchable compounds. In some embodiments, the exogenousantigen-expressing EHC does not comprise an activatable moleculardetection agent capable of producing a detectable response. In someembodiments, the exogenous antigen-expressing EHC does not comprise adiagnostic compound. In some embodiments, the exogenousantigen-expressing EHC does not comprise a virus or bacterium. In someembodiments, the exogenous antigen-expressing EHC is not generated fromor does not comprise an autologous CD34+ cell. In some embodiments,methods of treatment and prevention using exogenous antigen-expressingEHCs generated from erythroid cells described herein do not comprise thestep of detecting the erythroid cell in vivo, e.g., through a detectionagent that is associated with the erythroid cell. In some embodiments,the exogenous antigen-expressing EHC is not generated from a human donorpluripotent hematopoietic stem cell. In some embodiments, a populationof exogenous antigen-expressing EHCs is not expanded in a bioreactor. Insome embodiments, the population of exogenous antigen-expressing EHCsafter expansion and/or differentiation does not comprise a singlespecies of differentiated human blood cells. In some embodiments, theexogenous antigen-expressing EHC is not a differentiated, mature humanblood cell. In some embodiments, the exogenous antigen-expressing EHC isnot generated from a blood cell derived from a universal donor, e.g.blood type O, Rh factor negative. In some embodiments, an exogenous ADApolypeptide antigen-expressing EHC is not used to treat severe combinedimmune deficiency (ADA-SCID). In some embodiments, methods of expansionand differentiation of the exogenous antigen-expressing EHCs do notinclude culturing the exogenous antigen-expressing EHCs in a mediumcomprising a myeloproliferative receptor (mpl) ligand. In someembodiments, the exogenous antigen-expressing EHC does not comprise apayload comprising a synthetic triphosphorylated nucleoside analog. Insome embodiments, the exogenous antigen-expressing EHC does not comprisea payload comprising 2′,3′-dideoxycytidine-5′-triphosphate (ddCTP)and/or 3′-azido-3′-deoxythymidine-5′-triphosphate (AZT-TP). In someembodiments, the exogenous antigen-expressing EHC does not comprise apayload comprising a bisphosphonate. In some embodiments, the exogenousantigen-expressing EHC is generated by contacting an erythroid cell withan exogenous antigen and optionally a payload without lysing andresealing the cells to incorporate the exogenous antigen and/or payload.In some embodiments, the exogenous antigen-expressing EHC is generatedby contacting an erythroid cell with an exogenous antigen and optionallya payload, wherein contacting does not comprise hypotonic dialysis. Insome embodiments, the exogenous antigen-expressing EHC is generated bycontacting an erythroid cell with an exogenous antigen and optionally apayload, wherein contacting does not include loading the exogenousantigen and/or payload into or onto the erythroid cell. In someembodiments, the exogenous antigen is generated in an entity that is notthe erythroid cell to be contacted and/or the exogenous antigen isisolated from a sample that does not comprise the erythroid cell to becontacted. For example, for a polypeptide exogenous antigen suitableentities include a cell line, an in vitro expression system, a bacterialexpression system, etc.

In some embodiments, the exogenous antigen polypeptide expressed by theEHC is present on the surface of the EHC but is not non-covalently boundto the surface of the EHC. In some embodiments, the non-covalentattachment of the antigen to the surface of the EHC is not mediated byan antibody, an antibody-fragment, an antibody-like polypeptide, or anon-antibody polypeptide binding scaffold. In some embodiments, thenon-covalent attachment of the exogenous antigen to the surface of theEHC is not directed against an erythroid cell antigen such as Band 3(CD233), aquaporin-1, Glut-1, Kidd antigen, RhAg/Rh50 (CD241), Rh(CD240), Rh30 CE (CD240CE), Rh30D (CD240D), Kx, glycophorin B (CD235b),glycophorin C (CD235c), glycophorin D (CD235d), Kell (CD238),Duffy/DARCi (CD234), CR1 (CD35), DAF (CD55), globoside, CD44, ICAM-4(CD242), Lu/B-CAM (CD239), XG1/XG2 (CD99), EMMPRIN/neur. thelin (CD147),JMH, glycosyltransferase, Cartwright, Dombrock, C4A/CAB, Scianna, MER2,s tomatin, BA-1 (CD24), GPIV (CD36), CD108, CD139, and H antigen(CD173), or another erythrocyte-binding moiety.

In some embodiments, the exogenous antigen polypeptide is not generatedapart from the EHC and then conjugated to the EHC. In some embodiments,the exogenous antigen polypeptide is not enzymatically conjugated, e.g.through an autocatalytic isopeptide bond-forming reaction such ascarried out, e.g. by a transpeptidase, a sortase, and/or isopeptidase.In one embodiment, the exogenous antigen polypeptide is notenzymatically conjugated using a sortase.

In some embodiments, the exogenous antigen polypeptide is not chemicallyconjugated, e.g. through a cross-linking agent such as a carbodiimide(including sortase 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)).

In one embodiment, the exogenous antigen is not generated apart from theEHC and then encapsulated by the EHC. In one embodiment, theencapsulation of the exogenous antigen is not mediated by hypotonicdialysis of the EHC in the presence of exogenous antigen.

Many modifications and other embodiments of the inventions set forthherein will easily come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural references unless the contentclearly dictates otherwise.

The use of the alternative (e.g., “or”) should be understood to meaneither one, both, or any combination thereof of the alternatives.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

As used herein, any concentration range, percentage range, ratio range,or integer range is to be understood to include the value of any integerwithin the recited range and, when appropriate, fractions thereof (suchas one tenth and one hundredth of an integer), unless otherwiseindicated.

“Comprise,” “comprising,” and “comprises” and “comprised of” as usedherein are synonymous with “include”, “including”, “includes” or“contain”, “containing”, “contains” and are inclusive or open-endedterms that specifies the presence of what follows e.g. component and donot exclude or preclude the presence of additional, non-recitedcomponents, features, element, members, steps, known in the art ordisclosed therein.

As used herein, the terms “such as”, “for example” and the like areintended to refer to exemplary embodiments and not to limit the scope ofthe present disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, preferred materialsand methods are described herein.

All publications and patent applications cited in this specification areherein incorporated by reference in their entirety for all purposes asif each individual publication or patent application were specificallyand individually indicated to be incorporated by reference for allpurposes. The publications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the inventorsdescribed herein are not entitled to antedate such disclosure by virtueof prior invention or for any other reason.

Definitions

“Administration,” “administering” and variants thereof means introducinga composition, such as an exogenous antigen-expressing EHC, or agentinto a subject and includes concurrent and sequential introduction of acomposition or agent. The introduction of a composition or agent into asubject is by any suitable route, including orally, pulmonarily,intranasally, parenterally (intravenously, intramuscularly,intraperitoneally, or subcutaneously), rectally, intralymphatically, ortopically. Administration includes self-administration and theadministration by another. A suitable route of administration allows thecomposition or the agent to perform its intended function. For example,if a suitable route is intravenous, the composition is administered byintroducing the composition or agent into a vein of the subject.Administration can be carried out by any suitable route,

“Anchor” or “anchor domain” or “A domain” is used to refer to theportion of an exogenous antigen polypeptide, including a fusion orchimeric exogenous antigen polypeptide that is in contact with the cellmembrane of an EHC. The exogenous antigen polypeptide may interact withthe lipid cell membrane layer via a phospholipid tail insertion,covalent binding to a lipid layer constituent, an ionic bond, hydrogenbond, or via a single or multi-pass transmembrane polypeptide domainthat cross one or more of the lipid cell membrane layers.

As used herein, the term “antibody” encompasses an immunoglobulinwhether natural or partly or wholly synthetically produced, andfragments thereof. The term also covers any protein having a bindingdomain which is homologous to an immunoglobulin binding domain. Theseproteins can be derived from natural sources, or partly or whollysynthetically produced. “Antibody” further includes a polypeptidecomprising a framework region from an immunoglobulin gene or fragmentsthereof that specifically binds and recognizes an antigen. Use of theterm antibody is meant to include whole antibodies, polyclonal,monoclonal and recombinant antibodies, fragments thereof, and furtherincludes single-chain antibodies, humanized antibodies; murineantibodies; chimeric, mouse-human, mouse-primate, primate-humanmonoclonal antibodies, anti-idiotype antibodies, antibody fragments,such as, e.g., scFv, (scFv)2, Fab, Fab′, and F(ab′)2, F(ab1)2, Fv, dAb,and Fd fragments, diabodies, and antibody-related polypeptides. Antibodyincludes bispecific antibodies and multispecific antibodies so long asthey exhibit the desired biological activity or function.

The term “antigen binding fragment” used herein refers to fragments ofan intact immunoglobulin, and any part of a polypeptide includingantigen binding regions having the ability to specifically bind to theantigen. For example, the antigen binding fragment may be a F(ab′)2fragment, a Fab' fragment, a Fab fragment, a Fv fragment, or a scFvfragment, but is not limited thereto. A Fab fragment has one antigenbinding site and contains the variable regions of a light chain and aheavy chain, the constant region of the light chain, and the firstconstant region CH1 of the heavy chain. A Fab' fragment differs from aFab fragment in that the Fab′ fragment additionally includes the hingeregion of the heavy chain, including at least one cysteine residue atthe C-terminal of the heavy chain CH1 region. The F(ab′)2 fragment isproduced whereby cysteine residues of the Fab' fragment are joined by adisulfide bond at the hinge region. A Fv fragment is the minimalantibody fragment having only heavy chain variable regions and lightchain variable regions, and a recombinant technique for producing the Fvfragment is well known in the art. Two-chain Fv fragments may have astructure in which heavy chain variable regions are linked to lightchain variable regions by a non-covalent bond. Single-chain Fv (scFv)fragments generally may have a dimer structure as in the two-chain Fvfragments in which heavy chain variable regions are covalently bound tolight chain variable regions via a peptide linker or heavy and lightchain variable regions are directly linked to each other at theC-terminal thereof. The antigen binding fragment may be obtained using aprotease (for example, a whole antibody is digested with papain toobtain Fab fragments, and is digested with pepsin to obtain F(ab′)2fragments),and may be prepared by a genetic recombinant technique. A dAbfragment consists of a VH domain. Single-chain antibody molecules maycomprise a polymer with a number of individual molecules, for example,dimmer, trimer or other polymers.

“Applicator” refers to any device used to connect to a subject. Thisincludes, e.g., needles, cannulae, catheters, and tubing.

“Associated with” when used to describe the relationships among multiplecompounds or molecules encompasses such as, e.g., any interactionbetween an exogenous antigen and a target or between an exogenousantigen-expressing EHC and a target. This includes enzymaticinteraction, ionic binding, covalent binding, non-covalent binding,hydrogen bonding, London forces, van der Waals forces, hydrophobicinteraction, lipophilic interactions, magnetic interactions,electrostatic interactions, and the like.

“Associated with” when used to describe the relationships among atarget, entity, compound, agent, or molecule and a disease, disorder,condition, symptom or phenotype is any link that may reasonably be madebetween them, including a causal link, or a statistical significantlink, an empirically established link, a suggested link, whether or notcausative of the disease, disorder, condition, symptom or phenotype.

“Autoimmune disorders” generally are conditions in which a subject'simmune system attacks the body's own cells, causing tissue destruction.Autoimmune disorders may be diagnosed using blood tests, cerebrospinalfluid analysis, electromyogram (measures muscle function), and magneticresonance imaging of the brain, but antibody testing in the blood, forself-antibodies (or auto-antibodies) is particularly useful. Usually,IgG class antibodies are associated with autoimmune diseases.

“Binding” describes an interaction among compounds or molecules, e.g.,between an exogenous antigen and a target or between an exogenousantigen-expressing EHC and a target, that comes about by covalentbinding or non-covalent binding, including ionic binding, electrostaticinteractions, hydrogen bonding, London forces, van der Waals forces,hydrophobic interaction, lipophilic interactions, and similar.

The “biological activity of a polypeptide” refers to any molecularactivity or phenotype (such as, e.g., binding, signal transduction,catalytic, etc.) that is caused by the polypeptide, such as an exogenousantigen polypeptide.

As used herein, the term “biological sample” refers to any type ofmaterial of biological origin isolated from a subject, including, forexample, DNA, RNA, lipids, carbohydrates, and protein. The term“biological sample” includes tissues, cells and biological fluidsisolated from a subject. Biological samples include, e.g., but are notlimited to, whole blood, plasma, serum, semen, saliva, tears, urine,fecal material, sweat, buccal, skin, cerebrospinal fluid, bone marrow,bile, hair, muscle biopsy, organ tissue or other material of biologicalorigin known by those of ordinary skill in the art. Biological samplescan be obtained from, e.g., biopsies of internal organs or from cancers.Biological samples can be obtained from subjects for diagnosis orresearch or can be obtained from healthy subjects, as controls or forbasic research.

The “clearance rate” as used herein is calculated by measuring theamount or concentration of, e.g., exogenous antigen, target-exogenousantigen, or exogenous antigen-expressing EHCs remaining in thecirculatory system of a subject over time. For example, 1%, 2%, 3%, 4%,5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% oftarget detected in a first sample may still be detected in a secondsample that is taken 1 hour, 5 hours, 10 hours, 24 hours, 2 days, 3days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5years later. The clearance rate may alternatively be expressed as:number of entities (e.g., target/exogenous antigen) per unit of time(e.g., per day). An increase in clearance rate is a rate greater thanthat exhibited in an untreated or healthy suitable control subject. Adecrease in clearance rate is a rate less than that exhibited in anuntreated or healthy suitable control subject. The increase or decreasemay be 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 150%, 200%, 500%, 1000% or may be 1.1-fold, 1.2-fold, 1.3fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold,20-fold, 50-fold, 100-fold, 500-fold, or 1000-fold.

“Cleaving” as used herein is a process that disrupts a bondinginteraction present in a target, such as a polypeptide or nucleic e.g.,to produce two or more entities that after cleaving can be separatedfrom one another. The separation can involve, e.g., disrupt an ionicbond, a covalent bond, a polar covalent bond, a non-polar covalent bond,or a metallic bond. As cleaving applies to polypeptide targets, cleavagecan involve breaking one or more peptide bonds. As cleaving applies tosmall molecule targets, cleavage can involve breaking one or more carbonor sulfide bonds. As cleaving applies to nucleotide sequences, cleavagecan involve breaking one or more phosphodiester bonds. As cleavingapplies to microbes such as bacteria, fungi, or viruses, cleavage caninvolve lysis of a membrane or capsid structure. Cleaving can be carriedout by an enzyme, e.g., a catalytically active exogenous antigenpolypeptide. Exogenous antigens can comprise, e.g., exonuclease,endonuclease, or protease activity.

The “circulatory system of a subject,” as used herein, encompasses thespace occupied by whole blood and optionally the lymphatic system in ahuman, inclusive of plasma and all circulating cells and molecules, anddistributed throughout arteries, veins, capillaries, and lymphaticvessels of all tissues. The “circulatory concentration” is theconcentration of a target, e.g., a cell, polypeptide (such as anantibody, pathogenic antigen, etc.), therapeutic agent, small molecule,metabolite or other entity, an exogenous antigen or an exogenousantigen-expressing EHC in the space defined as the circulatory system.In certain embodiments, the concentration may be defined as the numberof free (unbound) entities in a given volume. In other embodiments, theconcentration may be defined as the total number of entities in a givenvolume.

The term “complementarity determining region (CDR)” used herein refersto an amino acid sequence found in the variable region of a heavy chainor a light chain of an immunoglobulin. The CDRs determine thespecificity of an antibody and may provide a contact residue for bindingto a specific epitope of an antigen. The heavy chain and the light chainmay respectively include three CDRs (CDRH1, CDRH2, and CDRH3, and CDRL1,CDRL2, and CDRL3). Four framework regions, which have more highlyconserved amino acid sequences than the CDRs, separate the CDR regionsin the VH or VL.

A “complex” as used herein comprises an association of two or moreentities. A complex may comprise one or more polypeptides, nucleic acid,lipids, carbohydrates, inorganic compounds, organic compounds, and thelike. A complex can be functional (multiunit polypeptides) ornon-functional (e.g., aggregates or precipitates) and may havebeneficial or detrimental properties (e.g., immune complexes). Complexesmay be naturally occurring or may be man-made or synthetic. Syntheticcomplexes include higher order entities, e.g., subcellular structuresand cells if they comprise a synthetic compound or molecule.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.As used herein the term “conservative amino acid substitution” isillustrated by a substitution among amino acids within each of thefollowing groups: (1) glycine, alanine, valine, leucine, and isoleucine,(2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine,(4) aspartate and glutamate, (5) glutamine and asparagine, and (6)lysine, arginine and histidine.

“Decrease,” in the context of a symptom of a treated disease, disorderor condition, refers to a reduction in measurable or conveyableparameters associated with the disease or condition that manifest assymptoms. Examples of measurable parameters are a reduction in thesubject's body temperature, a reduction in the concentration of targetsin a sample taken from the subject, reduction in the intensity ofinflammation or size of an inflamed area, reduction in the number ofinfiltrating cells, reduction in the number of episodes associated withthe disease, disorder or condition, increase/decrease in organ size,weight gain/loss, etc. Examples of conveyable parameters are, e.g., thesubject's own assessment of well being and quality of life. For example,for self-antibody mediated diseases, the decrease may be quantified asone, or a combination of, the following parameters: reducedinflammation, reduced flare-ups, reduced fatigue, reduced bloodclotting, reduced swelling, increased energy, or increased hair growth,etc. The parameters that may be quantified are those appropriate forassessing the specific disease, disorder or condition that is beingtreated. Delay, in the context of symptoms of a treated disease,disorder or condition, refers to the significant extension of amanageable health condition that would otherwise become exacerbated,using a treatment.

“Degrading” is defined as the process in which a target is eitherdirectly, or indirectly, reduced, inactivated, decomposed,deconstructed, lysed, dissolved, broken, lessened, impaired, weakened,deteriorated, diminished, or partitioned.

“Different polypeptide origin” refers to the organism or species fromwhich a genetic sequence encoding the polypeptide, the polypeptide, orportion thereof, is sourced. In certain embodiments, a fusion comprisingpolypeptides of different polypeptide origin may include an exogenousantigen polypeptide that is encoded by the genetic sequence for humanadenosine deaminase and the genetic sequence for phenylalaninehydroxylase from chromobacterium violaceum.

A “domain” is a part of a polypeptide, such as an exogenous antigenpolypeptide that is generally having a 3-dimensional structure and mayexhibit a distinct activity, function, such as, e.g., a catalytic, anenzymatic, a structural role, or a binding function.

By an “enriched population of cells” it is meant a population of cellsthat is substantially comprised of a particular cell of interest. In anenriched population, 50% or more of the cells in the population are thecells of interest, e.g., 50%, 60%, 70%, usually 80%, 85%, 90%, moreusually 92%, 95%, 96%, 97%, 98%, or 99%, sometimes as much as 100% ofthe cells in the population. The separation of cells of interest from acomplex mixture or heterogeneous culture of cells may be performed byany convenient means known in the art, for example, by affinityseparation techniques such as magnetic separation using magnetic beadscoated with an affinity reagent, affinity chromatography, or “panning”with an affinity reagent attached to a solid matrix, e.g., plate, orother convenient technique. Other techniques providing accurateseparation include fluorescence activated cell sorters, which can havevarying degrees of sophistication, such as multiple color channels, lowangle and obtuse light scattering detecting channels, impedancechannels, etc. The cells may be selected against dead cells by employingdyes associated with dead cells. Any technique may be employed which isnot unduly detrimental to the viability of the desired cells.

“Enucleation” is the rendering of a cell to a non-replicative state,either through inactivation or removal of the nucleus.

An “epitope” includes any segment on an antigen to which an antibody orother ligand or binding molecule binds. An epitope may consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics. In someembodiments, exogenous antigens comprise specific epitopes. In someembodiments, targets comprise specific epitopes.

“Erythroid cells” as used herein, include nucleated red blood cells, redblood cell precursors, and enucleated red blood cells and those listedin Table Al. For example, the erythroid cells are a cord blood stemcell, a CD34+ cell, a hematopoietic stem cell (HSC), a spleen colonyforming (CFU-S) cell, a common myeloid progenitor (CMP) cell, ablastocyte colony-forming cell, a burst forming unit-erythroid (BFU-E),a megakaryocyte-erythroid progenitor (MEP) cell, an erythroidcolony-forming unit (CFU-E), a reticulocyte, an erythrocyte, an inducedpluripotent stem cell (iPSC), a mesenchymal stem cell (MSC), apolychromatic normoblast, an orthochromatic normoblast, or a combinationthereof. In some embodiments, the erythroid cells are immortal orimmortalized cells. For example, immortalized erythroblast cells can begenerated by retroviral transduction of CD34+ hematopoietic progenitorcells to express Oct4, Sox2, Klf4, cMyc, and suppress TP53 (e.g., asdescribed in Huang et al., Mol Ther 2013, epub ahead of print September3). In addition, the cells may be intended for autologous use or providea source for allogeneic transfusion. Erythroid cells can be contactedwith an exogenous antigen to generate an exogenous antigen-expressingEHC. Erythroid cells comprising an exogenous antigen are one example ofan exogenous antigen-expressing EHC. In some embodiments, erythroidcells are cultured. In some embodiments, erythroid progenitor cells arecontacted with an exogenous antigen to generate an exogenousantigen-expressing EHC.

As used herein, the term “thromboid cell” refers to a cell of the stemcell-megakaryocyte-platelet lineage, including for examplemegakayrocytes and platelets, or cells that are induced to differentiateby thrombopoietin, or cells that express surface markers associated withthis lineage, for example CD41 (GP IIb/IIIa), CD42a (GPIX), CD42b(GPIb), and CD61 (avb3, vitronectin receptor), PAC-1 (activatedIIb/IIIa), CD62P (P-selectin), CD31 (PECAM) and CD63.

As used herein, the term “enucleated hematopoietic cell” (EHC) refers toa hematopoietic cell, human or non-human, that is or has been renderedenucleated as defined herein. This definition encompasses the both“erythroid cells” and “thromboid cells” as defined herein.

As used herein, the term “excipient” refers to an inert substance addedto a pharmaceutical composition to further facilitate administration ofa compound. Examples of excipients include, but are not limited to,calcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils, anti-coagulants,and polyethylene glycols.

The term “exogenous” as used herein means a cellular component orfunction that is generated by a carbohydrate, polysaccharide, lipid,oligonucleotide or polypeptide not found naturally within a cell or theenhancement or manipulation of a cellular component or function that isendogenous to a cell, including, e.g., a fusion protein comprising anexogenous polypeptide antigen and an endogenous protein or a functionalfragment thereof. The exogenous antigen, including an exogenous antigenpolypeptide is “exogenous” or “heterologous”, thus it may either notnaturally exist, such as a fusion or chimera comprising domains ofdifferent polypeptide or species origin, it may not naturally occur in anaturally occurring cell, such as an unmodified erythrocyte or platelet,it may not function in the same way as a naturally occurring polypeptidewould, or it may not naturally occur in the quantity that the exogenousantigen polypeptide occurs, e.g., in embodiments in which the exogenousantigen is overexpressed as compared to the expression of a naturallyoccurring polypeptide in an unmodified cell. In some embodiments, thepolypeptide exogenous antigen is expressed from an exogenous nucleicacid. In some embodiments, the exogenous antigen is isolated from asource and loaded into or conjugated to an exogenous antigen-expressingEHC. The term “exogenous” when used in the context of nucleic acidincludes a transgene and recombinant nucleic acids.

As used herein, the term “expression” or “expressing” refers to theprocess to produce a polypeptide, such as an exogenous antigenpolypeptide including transcription and translation. Expression may be,e.g., increased by a number of approaches, including: increasing thenumber of genes encoding the polypeptide, increasing the transcriptionof the gene (such as by placing the gene under the control of aconstitutive promoter), increasing the translation of the gene, knockingout of a competitive gene, or a combination of these and/or otherapproaches. The term “expression” or “expressing” also include EHCs thatcomprise an exogenous polypeptide that was at one time activelyexpressed by the EHC but active expression (defined as transcription andtranslation) since has ceased. For example, the exogenous antigenpolypeptide was actively expressed (i.e. transcribed and translated) byan EHC prior to the enucleation event and the antigen polypeptide isretained by the EHC after enucleation but no longer actively expressed,e.g. for lack of encoding nucleic acid. For example, the EHC maycomprise an exogenous antigen polypeptide encoded by an exogenousnucleic acid. During enucleation the exogenous antigen polypeptide isretained by the EHC whereas the exogenous nucleic acid is not retained,such EHC is said to be “antigen-expressing” or “expressing antigen” evenin the event that the active expression (transcription and translation)of the antigen polypeptide is effectively terminated and/or the EHC doesnot contain a substantial amount of a replicating nucleic acid.

A “functional” exogenous antigen or exogenous antigen-expressing EHCexhibits a desired or specified activity or characteristic, includingenzymatic, catalytic or metabolic activity, structural integrity,immunogenic complementarity, target binding, and correct localization oris capable of promoting a desired or specified effect or phenotype.

“Fusion or chimera” is a polypeptide sequence, or corresponding encodingnucleotide sequence, that is derived from the combination of two or moresequences that are not found together in nature. This may be acombination of separate sequences derived from separate genes within thesame genome, or from heterologous genes derived from distinctlydifferent species' genomes.

“Genetic material” refers to nucleic acid molecules having nucleotidesequences of adenosine, thymine, uracil, cytosine, and guanine capableof encoding a gene.

The term “heavy chain” used herein is understood to include afull-length heavy chain including a variable region (VH) having aminoacid sequences that determine specificity for antigens and a constantregion having three constant domains (CH1, CH2, and CH3), and fragmentsthereof. In addition, the term “light chain” used herein is understoodto include a full-length light chain including a variable region (VL)having amino acid sequences that determine specificity for antigens anda constant region (CL), and fragments thereof.

The term “homolog” indicates polypeptides, including exogenous antigenpolypeptide that have the same or conserved residues at a correspondingposition in their primary, secondary or tertiary structure. Functionalhomologs include exogenous antigens and other polypeptides that exhibitsimilar function and/or specificity (e.g., for a particular target).

A naturally occurring intact antibody, or immunoglobulin, includes fourpolypeptides: two full-length light chains and two full-length heavychains, in which each light chain is linked to a heavy chain bydisulfide bonds. Each heavy chain has a constant region and a variableregion. Similarly, each light chain has a constant region and a variableregion. There are five heavy chain classes (isotypes): gamma (γ), mu(μ), alpha (α), delta (δ), or epsilon (ε), and additionally severalsubclasses gamma 1 (γ1), gamma 2(γ2), gamma 3(γ3), gamma 4(γ4), alpha1(α1), and alpha 2(α2). The light chain constant region can be eitherkappa (κ) or lambda (λ) type. The variable regions differ in sequenceamong antibodies and are used in the binding and specificity of a givenantibody to its particular antigen.

As used herein, the term “increase,” “enhance,” “stimulate,” and/or“induce” (and like terms) generally refers to the act of improving orincreasing, either directly or indirectly, a concentration, level,function, activity, or behavior relative to the natural, expected, oraverage, or relative to a control condition.

As used herein, the term “inhibit,” “suppress,” “decrease,” “interfere,”and/or “reduce” (and like terms) generally refers to the act ofreducing, either directly or indirectly, a concentration, level,function, activity, or behavior relative to the natural, expected, oraverage, or relative to a control condition.

A “library” as used herein includes a collection of nucleic acidmolecules (e.g., DNA, RNA) having diverse nucleic acid sequences, agenetically diverse collection of clones, a collection of diversepolypeptides, a diverse collection of cells, such as EHCs, etc.

As used herein, “a mammalian subject” includes all mammals, includingwithout limitation, humans, domestic animals (e.g., dogs, cats and thelike), farm animals (e.g., cows, sheep, pigs, horses and the like) andlaboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigs andthe like). The terms “individual,” “subject,” “host,” and “patient,” areused interchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, particularly humans. Themethods described herein are applicable to both human therapy andveterinary applications. In some embodiments, the subject is a mammal,and in other embodiments the subject is a human.

“Medical device” refers to any device, apparatus or machine used todeliver a dose of an exogenous antigen-expressing EHC and/or atherapeutic agent. This includes containers, bottles, vials, syringes,bags, cartridges, cassettes, magazines, cylinders, or canisters.

“Medical kit” refers to a packaged unit that includes a medical deviceor applicator, an appropriate dosage of exogenous antigen-expressingEHC, optionally including a therapeutic agent, and relevant labeling andinstructions.

As used herein, the term “modulate,” “modulating”, “modify,” and/or“modulator” generally refers to the ability to alter, by increase ordecrease, e.g., directly or indirectlypromoting/stimulating/upregulating or interferingwith/inhibiting/downregulating a specific concentration, level,expression, function or behavior, such as, e.g., to act as an antagonistor agonist. In some instances a modulator may increase and/or decrease acertain concentration, level, activity or function relative to acontrol, or relative to the average level of activity that wouldgenerally be expected or relative to a control level of activity.

“Membrane” as used herein is a boundary layer that separates an interiorspace from an exterior space comprising one or more biologicalcompounds, typically lipids, and optionally polypeptides. Membranes canbe lipid bilayers. In certain embodiments, membranes comprise one ormore of phosphatidylcholine, sphingomyelin, lysophosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, orphosphatidic acid. In some embodiments, membranes comprise one or morepolypeptides such as ankyrin and coenzyme Q10. Included in thedefinition of membrane are cell membranes comprising, e.g., aphospholipid bilayer and cell membrane associated polypeptides.

The phrase “nucleic acid molecule” refers to a single or double-strandedpolymer of deoxyribonucleotide or ribonucleotide bases. It includeschromosomal DNA and self-replicating plasmids, vectors, mRNA, tRNA,siRNA, etc. which may be recombinant and from which exogenouspolypeptides may be expressed when the nucleic acid is introduced into acell.

Orthologs are defined as genes in different species that evolved from acommon ancestral gene by speciation.

The term “pharmaceutically-acceptable” and grammatical variationsthereof, refers to compositions, carriers, diluents and reagents capableof administration to or upon a subject without the production ofundesirable physiological effects to a degree that would prohibitadministration of the composition. For example,“pharmaceutically-acceptable excipient” includes an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients can be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans, as well as any carrier or diluent that does not causesignificant irritation to a subject and does not abrogate the biologicalactivity and properties of the administered compound.

Some agents may be administered as “pharmaceutically acceptable salt”,e.g., prepared from inorganic and organic acids. Salts derived frominorganic acids include hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid, and the like. Salts derived fromorganic acids include acetic acid, propionic acid, glycolic acid,pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid,maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluene-sulfonic acid, salicylic acid, and the like. Salts can also beprepared from inorganic and organic bases. Salts derived from inorganicbases, include by way of example only, sodium, potassium, lithium,ammonium, calcium and magnesium salts. Salts derived from organic basesinclude, but are not limited to, salts of primary, secondary andtertiary amines Any ordinary skilled person in the art will know how toselect a proper pharmaceutically acceptable carrier, a pharmaceuticallyacceptable salt thereof for implementing this invention without undueexperimentation.

As used herein, the term “pharmaceutical composition” refers to one ormore of the compounds described herein, such as, e.g., an exogenousantigen-expressing EHC mixed or intermingled with, or suspended in oneor more other chemical components, such as physiologically acceptablecarriers and excipients. One purpose of a pharmaceutical composition isto facilitate administration of a compound to a subject.

Certain embodiments provide various polypeptide molecules havingsequences associated with a desired function or activity, such asexogenous antigen polypeptides. A polypeptide is a term that refers to achain of amino acid residues, regardless of post-translationalmodification (e.g., phosphorylation or glycosylation) and/orcomplexation with additional polypeptides, synthesis into multisubunitcomplexes, with nucleic acids and/or carbohydrates, or other molecules.Proteoglycans therefore also are referred to herein as polypeptides. Incertain embodiments, the exogenous antigen-expressing EHC comprises apolypeptide exogenous antigen. In certain embodiments, the exogenousantigen-expressing EHC comprises one or more non-exogenous antigenpolypeptides that are optionally membrane-associated.

The term “pharmaceutically active agent” or “pharmaceutical agent” isdefined as any compound, e.g., a small molecule drug, or a biologic(e.g., a polypeptide drug or a nucleic acid drug) that when administeredto a subject has a measurable or conveyable effect on the subject, e.g.,it alleviates or decreases a symptom of a disease, disorder orcondition. In some embodiments, the pharmaceutical agent may beadministered prior to, in combination with, or following the delivery ofan exogenous antigen-expressing EHC. In some embodiments, thepharmaceutically active agent exerts a synergistic treatment effect withthe exogenous antigen-expressing EHC. In some embodiments, thepharmaceutically active agent exerts an additive treatment effect withthe exogenous antigen-expressing EHC.

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of an operably linked nucleic acid. Promotersinclude necessary nucleic acid sequences near the start site oftranscription. A promoter also optionally includes distal enhancer orrepressor elements. A “constitutive” promoter is a promoter that isactive under most environmental and developmental conditions. An“inducible” promoter is a promoter that is active under environmental ordevelopmental regulation. The term “operably linked” refers to afunctional linkage between a nucleic acid expression control sequence(such as a promoter, or array of transcription factor binding sites) anda second nucleic acid sequence, wherein the expression control sequencedirects transcription of the nucleic acid corresponding to the secondsequence.

A “exogenous antigen,” as used herein, is an entity capable ofinteracting with a target, e.g., to associate with or bind to a target.An exogenous antigen can comprise or can consist essentially of apolypeptide. In some embodiments, the exogenous antigen comprises apolypeptide, a carbohydrate, a nucleic acid, a lipid, a small molecule,or a combination thereof. In embodiments in which an exogenous antigenis a naturally occurring compound or molecule, the antigen is“exogenous” in the sense that it is an exogenous or heterologouscompound or molecule with regard to its presence in the EHC. In otherembodiments the antigen is “exogenous” in the sense that it is aman-made compound or molecule, such as a fusion or chimera, anon-naturally occurring polypeptide, carbohydrate, nucleic acid, lipid,or combination thereof, or a man-made small molecule or othertherapeutic agent. For example, the exogenous antigen may comprise afusion or chimera comprising one or more of an S domain, an A domain anda U domain. The S domain is a surface domain exposed to the environmentaround the EHC, such as the circulatory system of a subject. The Adomain is an anchor domain that attaches the S domain to the cellmembrane of the EHC. The U domain faces the unexposed side of or islocated within (i.e. in the intracellular space of) the EHC.Irrespective of any domains, an exogenous antigen may be located on thesurface of the exogenous antigen-expressing EHC or may be located withinthe EHC. The exogenous antigen may be associated with the membrane ofthe exogenous antigen-expressing EHC, e.g., the exogenous antigen isanchored in, conjugated to or otherwise bound to the membrane. In someembodiments, the exogenous antigen may be conjugated to the membrane ofthe exogenous antigen-expressing EHC by chemical or enzymaticconjugation. In other embodiments, the exogenous antigen is notconjugated to the membrane. In some embodiments, the exogenous antigenis not associated with the membrane of the exogenous antigen-expressingEHC and is located within the membrane-encapsulated intracellular spaceof the EHC. In some embodiments, an exogenous antigen located within theintracellular space of the EHC does not substantially diffuse out of theEHC and/or may not permeate the membrane. In other embodiments, theexogenous antigen may substantially diffuse out of the EHC and/or maypermeate the membrane. In some embodiments, the exogenous antigen isloaded, e.g., introduced into or put onto the EHC. An exogenous antigenthat is loaded is not biologically synthesized by the exogenousantigen-expressing EHC. An exogenous antigen suitable for loading may bee.g., produced in a cell-based expression system, isolated from abiological sample, chemically or enzymatically synthesized, and thenloaded into or onto the EHC. In some embodiments, the exogenous antigenmay be further modified by the exogenous antigen-expressing EHC afterloading. In other embodiments, the exogenous antigen is not modifiedafter loading. In some embodiments, the exogenous antigen polypeptide isnot loaded onto or into the EHC. In some embodiments, the exogenousantigen is made, e.g., biologically synthesized by the exogenousantigen-expressing EHC. Typically an exogenous antigen polypeptide isexpressed by the exogenous antigen-expressing EHC from an exogenousnucleic acid molecule (e.g., a DNA or mRNA) that was introduced into theEHC. The exogenous antigen may have a biological function that isretained when the antigen is expressed on the EHC. The exogenous antigenmay bind to and/or sequester a target. Alternatively or in addition theexogenous antigen may exhibit a catalytic activity toward the target,e.g., the exogenous antigen may convert or modify the target, or maydegrade the target. A product may then optionally be released from theexogenous antigen.

“Residency” of an exogenous antigen-expressing EHC refers to the periodof time it spends in a physiological location. The specific location ofthe exogenous antigen-expressing EHC may change during its lifetime and“residency” applies to the period of time spent in various environments,including vascular circulation, peripheral tissues, capillaries,digestive system, pulmonary system, nasal tissues, epidermal surface,and interstitial tissue. In specific embodiments, the exogenousantigen-expressing EHC resides in the circulatory system of a subject.

“Replicating nucleic acid” refers to deoxyribonucleic acid (DNA) that iscapable of being copied by enzymes dedicated to the increasing thenumber of copies of the DNA. Usually, DNA replication leads to theproduction of two identical replicas from one original DNA molecule. DNAreplication comprises the incorporation of nucleotides into a growingDNA strand by DNA polymerase matched to the template strand one at atime via the creation of phosphodiester bonds.

“Sequestering” is defined as cloistering, occluding, separating,segregating, hiding, insulating, or isolating of a target and preventingit from freely interacting with its environment.

“Specifically binding” or “specifically interacting”, as used herein,describes any interaction between two entities (e.g., a target with anexogenous antigen, such as an antibody with an antigen, a receptor witha ligand, an enzyme with a substrate, biotin with avidin, etc.) that issaturable, often reversible and so competitive, as these terms areunderstood by those of ordinary skill in the chemical and biochemicalarts. e.g., Specific binding involving biological molecules such as,e.g., proteins, peptides and nucleic acid occurs when one member of thebinding pair has a site with a shape and distribution of charged, polar,or hydrophobic moieties such that the interaction of the cognate ligandwith that site is characterized by favorable energetics (i.e., anegative free energy of binding). The specificity of the interaction maybe measured or expressed as a binding constant (Kd). The Kd may rangefrom a mM range to a fM range, including pM ranges, μM ranges and nMranges. Typical Kd values are below about 10⁻⁶ M, below about 10⁻⁷ M,below about 10⁻⁸ M, and in some embodiments below about 10⁻⁹ M.

As used herein, the term “substantially” or “substantial” refers, e.g.,to the presence, level, or concentration of an entity in a particularspace, the effect of one entity on another entity, or the effect of atreatment. For example, an activity, level or concentration of an entityis substantially increased if the increase is 2-fold, 3-fold, 4-fold,5-fold, 10-fold, 50-fold, 100-fold, or 1000-fold relative to a baseline.An activity, level or concentration of an entity is also substantiallyincreased if the increase is 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 200%, or 500% relative to a baseline. An entity may besubstantially present in a particular space if it can be detected bymethods known in the art. An entity may not be substantially present ina particular space if it is present at levels below the limit ofdetection for assays and methods known in the art. In some embodiments,an entity may not be substantially present in a particular space if itis barely detectable but only in non-functional quantities or minutequantities that do not cause or change a phenotype. In otherembodiments, an entity may not be substantially present in a particularpopulation if it is present and can be detected only in a small numberof constituents making up the population, e.g., less than 10%, 9%, 8%,7%, 6%, 5%, 4%, 3% 2% or less than 1%, 0.5%, 0.1% of constituents of thepopulation. For example, an exogenous nucleic acid may not be retainedupon enucleation, the cell is rendered non-replicative, and theenucleated cell is incapable of continued expression of the exogenousantigen polypeptide encoded by the exogenous nucleic acid. The loss ofthe ability of the cell to continue to significantly translate theexogenous polypeptide “effectively terminates” protein expression. Incertain embodiments, the exogenous antigen-expressing EHC issubstantially incapable of self-replication, e.g., the replication ofnucleic acids. For example, the exogenous antigen-expressing EHC doesnot substantially incorporate a nucleoside if contacted with labelednucleoside, such as thymidine, in an incorporation assay. In someembodiments, the exogenous antigen-expressing EHC does not contain asubstantial amount of self-replicating nucleic acids. The term“substantial identity” of polynucleotide or nucleic acid sequences meansthat a polynucleotide comprises a sequence that has at least 25%sequence identity. Alternatively, percent identity can be any integerfrom 25% to 100%. More preferred embodiments include at least: 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%compared to a reference sequence using the programs described herein;preferably BLAST using standard parameters.

“Synthetic” refers to a compound or molecule that is either man-made andnon-naturally occurring, or if it is naturally occurring is placed in acontext or location that it would not naturally exist, or if itnaturally exists in the context or location is in a state of purity, oris present in an amount, concentration or number that it would notnaturally be present in the context or location. Synthetic entities canbe isolated or purified compounds that are optionally chemically orenzymatically modified from their natural state, exogenous nucleicacids, exogenous (heterologous) exogenous antigens, and the like. Thepresence of a synthetic compound or molecule, as defined herein, in anyentity renders the entire entity “synthetic”. For example, a cellcomprising an exogenous antigen is a synthetic cell.

A “target,” as used herein, is an entity capable of interacting with anexogenous antigen, e.g., to associate with or bind to an exogenousantigen. A “target” includes, but is not limited to a polypeptide (e.g.,an antibody or antibody-related polypeptide, a complement constituent,an amyloid protein, a pathogen, a toxin, a prion), a molecule (e.g., ametabolite, a steroid, a hormone, a carbohydrate; an oligosaccharide; achemical; a polysaccharide, a DNA; an RNA; a lipid, an amino acid, anelement, a toxin or pathogen), a complex (e.g., an immune complex), or acell (e.g., a cancer cell, a macrophage, a bacterium, a fungus, a virus,or a parasite). A target is intended to be detected, diagnosed,impaired, destroyed or altered (e.g., functionally complemented) by themethods provided herein. The specific target may occur free or isassociated with other entities in the circulatory system of a subject.

A “target self-antibody,” as used herein, is a self-antibody associatedwith an autoimmune disease. Such self-antibodies may be detected andanalyzed using antibody binding tests involving contacting the subject'santibodies to samples of the subject's own tissue, usually thyroid,stomach, liver, and kidney tissue. Antibodies binding to the “self”tissue (comprising self-antigens) indicate an autoimmune disorder.

“Transgene” or “exogenous nucleic acid” refers to a foreign or nativenucleotide sequence that is introduced into an EHC. Transgene andexogenous nucleic acid are used interchangeably herein and encompassrecombinant nucleic acids.

As used herein, “treat,” “treating,” and/or “treatment” are an approachfor obtaining beneficial or desired clinical results, pharmacologicand/or physiologic effect, e.g., alleviation of the symptoms, preventingor eliminating said symptoms, and refer to both therapeutic treatmentand prophylactic or preventative treatment of the specific disease,disorder or condition. Beneficial or desired clinical results,pharmacologic and/or physiologic effect include, but are not limited to,preventing the disease, disorder or condition from occurring in asubject that may be predisposed to the disease, disorder or conditionbut does not yet experience or exhibit symptoms of the disease(prophylactic treatment), alleviation of symptoms of the disease,disorder or condition, diminishment of extent of the disease, disorderor condition, stabilization (i.e., not worsening) of the disease,disorder or condition, preventing spread of the disease, disorder orcondition, delaying or slowing of the disease, disorder or conditionprogression, amelioration or palliation of the disease, disorder orcondition, and combinations thereof, as well as prolonging survival ascompared to expected survival if not receiving treatment.

A “therapeutic agent” or “therapeutic molecule” includes a compound ormolecule that, when present in an effective amount, produces a desiredtherapeutic effect, pharmacologic and/or physiologic effect on a subjectin need thereof.

The term “therapeutically effective amount” or “effective amount” is anamount of an agent being administered to a subject sufficient to effectbeneficial or desired clinical results, pharmacologic and/or physiologiceffects. An effective amount can be administered in one or moreadministrations. An effective amount is typically sufficient topalliate, ameliorate, stabilize, reverse, slow or delay the progressionof the disease state. The effective amount thus refers to a quantity ofan agent or frequency of administration of a specific quantity of anagent sufficient to reasonably achieve a desired therapeutic and/orprophylactic effect. For example, it may include an amount that resultsin the prevention of, treatment of, or a decrease in, the symptomsassociated with a disease or condition that is being treated, e.g., thedisease or medical conditions associated with autoimmune response,overactive immune activation, or inhibitory antibody generation forwhich immune tolerance is desired, or the diseases or medical conditionsassociated with a target polypeptide. The amount of a therapeuticcomposition administered to the subject will depend on the type andseverity of the disease and on the characteristics of the individual,such as general health, pathologic conditions, diets, age, sex, bodyweight and tolerance to drugs. It will also depend on the degree,severity and type of disease. Further, the effective amount will dependon the methods of formulation and administration used, e.g.,administration time, administration route, excretion speed, and reactionsensitivity. The skilled artisan will be able to determine appropriatedosages depending on these and other factors. The compositions can alsobe administered in combination with one or more additional therapeuticcompounds. A desirable dosage of the pharmaceutical composition may bein the range of about 0.001 to 100 mg/kg for an adult. In one example,an intravenous administration is initiated at a dose which is minimallyeffective, and the dose is increased over a pre-selected time courseuntil a positive effect is observed. Subsequently, incremental increasesin dosage are made limiting to levels that produce a correspondingincrease in effect while taking into account any adverse affects thatmay appear. Non-limited examples of suitable dosages can range, forexample, from 1×10¹⁰ to 1×10¹⁴, from 1×10¹¹ to 1×10¹³, or from 5×10¹¹ to5×10¹² exogenous antigen-expressing EHCs of the present invention.Specific examples include about 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰,1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹,1×10¹², or more exogenous antigen-expressing EHCs of the presentinvention. Each dose of exogenous antigen-expressing EHCs can beadministered at intervals such as once daily, once weekly, twice weekly,once monthly, or twice monthly.

“Unbound” refers to the state of a target with which the exogenousantigen is capable of interacting. An unbound target is not associatedwith another entity or an exogenous antigen. An unbound exogenousantigen is not associated with another entity or a target. A target isconsidered “bound” once it is associated with the exogenous antigen oranother entity. Unbound targets include soluble forms of the target incirculation. Bound targets include targets that are embedded, associatedwith, linked to, or otherwise interacting with entities in circulationor peripheral tissue. Entities with which a target may interact includecirculating cells, peripheral endothelial tissue, immune complexes,glycolipids, microbes, immunoglobulins, serum albumin, clotting factors,lipoproteins, and electrolytes.

A “variant” is a polypeptide which differs from the original protein byone or more amino acid substitutions, deletions, insertions, or othermodifications. These modifications do not significantly change thebiological activity of the original protein. In many cases, a variantretains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or100% of the biological activity of original protein. The biologicalactivity of a variant can also be higher than that of the originalprotein. A variant can be naturally-occurring, such as by allelicvariation or polymorphism, or be deliberately engineered.

The amino acid sequence of a variant is substantially identical to thatof the original protein. In many embodiments, a variant shares at least50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more global sequence identityor similarity with the original protein. Sequence identity or similaritycan be determined using various methods known in the art, such as BasicLocal Alignment Tool (BLAST), dot matrix analysis, or the dynamicprogramming method. In one example, the sequence identity or similarityis determined by using the Genetics Computer Group (GCG) programs GAP(Needleman-Wunsch algorithm). The amino acid sequences of a variant andthe original protein can be substantially identical in one or moreregions, but divergent in other regions.

As used herein, the term “vector” is a nucleic acid molecule, preferablyself-replicating, which transfers and/or replicates an inserted nucleicacid molecule, such as a transgene or exogenous nucleic acid into and/orbetween host cells. It includes a plasmid or viral chromosome into whosegenome a fragment of recombinant DNA is inserted and used to introducerecombinant DNA, or a transgene, into an EHC.

EXAMPLES

The following examples are offered by way of illustration and not by wayof limitation.

Example 1 Culture of Erythroid Cells with Heterologous Gene Expression

CD34 cells are isolated from peripheral blood by supermagnetic microbeadselection by the use of Mini-MACS columns (Miltenyi Biotec; 94%+/−3%purity). The cells are cultured in erythroid differentiation medium(EDM) on the basis of IMDM supplemented with stabilized glutamine, 330ug/mL holo-human transferrin, 10 ug/mL recombinant human insulin, 2IU/mL heparin, and 5% solvent/detergent virus-inactivated plasma. Theexpansion procedure comprises 3 steps. In the first step (day 0 to day7), 10{circumflex over ( )}4/mL CD34 cells are cultured in EDM in thepresence of 1 uM hydrocortisone, 100 ng/mL SCF, 5 ng/mL IL-3, and 3IU/mL Epo. On day 4, 1 volume of cell culture is diluted in 4 volumes offresh medium containing SCF, IL-3, Epo, and hydrocortisone. In thesecond step (day 7 to day 11), the cells are resuspended at10{circumflex over ( )}5/mL in EDM supplemented with SCF and Epo. In thethird step (day 11 to day 18), the cells are cultured in EDMsupplemented with Epo alone. Cell counts are adjusted to7.5×10{circumflex over ( )}5 to 1×10{circumflex over ( )}6 and5-10×10{circumflex over ( )}6 cells/mL on days 11 and 15, respectively.Beyond day 18, the culture medium containing Epo is renewed twice aweek. The cultures are maintained at 37° C. in 5% CO2 in air.

The coding sequence of the antigen of interest is placed under thecontrol of an erythroid-specific promoter, e.g. GATA-1, and terminatedwith a poly-A tail, see e.g. Repik et al., Clin Exp Immunol 2005,140:230. This sequence is encoded in a lentiviral vector (e.g. EF1,System Biosciences, Inc.). The vector is produced by standard methodsfrom 293T cells. The lentiviral vector is transduced into humanhematopoietic progenitor cell, e.g. CD34+ cell or immortalizederythroblast or iPS cell, for example as described by Chang et al., NatBiotechnol 2006, 24:1017, during days 1-4 of culturing. Subsequentexpansion and differentiation stages are performed as described above.

Example 2 Loading of Protein into Erythroid Cell by Hypotonic/HypertonicCycling

Antigen, in this case OVA, is added to a RBC suspension at a finalconcentration of 0.5 or 5 mg/ml with a hematocrit (Hct) of 70%. After ahypotonic dialysis process (50 mOsmol/kg), RBC are resealed by adding ahypertonic solution (1900 mOsmol/kg) at 37 C for 30 min. OVA-loaded RBCare washed four times with a 0.9% NaCl+0.2% glucose solution,centrifuged at 1000×g for 10 min at 4 C and adjusted to a Hct of 50%with plasma or buffer.

Example 3 Cell Surface Labeling with Heterobifunctional Crosslinker

Antigen with exposed, reduced thiol group (antigen-SH) is prepared byincubation with 5 mM TCEP. The reducing agent is removed by sizeexclusion chromatography prior to conjugation. Cells are incubated withmaleimide-PEG-NHS crosslinker, e.g. SM(PEG)_(x) (Thermo Scientific) for30 minutes. The NHS group reacts with free amine groups on cell surfaceantigens. Reacted cells are washed in PBS to remove excess crosslinker.Antigen-SH solution is introduced to the cells-plus-crosslinkerssolution, and the maleimide-SH reaction is allowed to proceed for 30minutes. Cells are pelleted and washed to remove unbound antigen.

Example 4 Cell Surface Labeling with Sortase

Cells expressing surface proteinst that contain the Sortase A acceptorsequence, LPXTG, are incubated with 100 uM sortase A in a reactionbuffer consisting of 50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM CaCl₂, for4 h at 37° C. 10x molar excess of nucleophile, GGG-antigen, isintroduced and the reaction is allowed to proceed at room temperatureovernight. Following the reaction, excess sortase and unreactednucleophile is removed by pelleting and washing the cells.

Example 5 Cell Surface Labeling with Click Chemistry

Cells are reacted with Alkyne-NHS Ester to conjugate the alkyne group toexposed primary amines on the cell surface, per manufacturer'sinstructions (e.g. Glen Research). Excess alkyne-NHS ester is removed bypelleting and washing the cells. Antigen is reacted with Azide-NHS esterto conjugate the azide group to exposed primary amines on the antigen,per manufacturer's instructions (e.g. Thermo Scientific). Excessazide-NHS ester is removed by size exclusion chromatography.Alkyne-cells and azide-antigen are reacted by copper-catalyzedcycloaddition at room temperature.

Example 6 Measurement of T Cell Proliferation in a Mouse

CD8⁺ T cells from OTI (CD45.2⁺) mouse spleens are isolated using a CD8magnetic bead negative selection kit (Miltenyi Biotec) as per themanufacturer's instructions. Freshly isolated CD8⁺ OTI cells areresuspended in PBS and labeled with 1 μM CFSE (Invitrogen) for 6 min atroom temperature, and the reaction is quenched for 1 min with an equalvolume of Iscove's modified Dulbecco's medium (IMDM) with 10% (vol/vol)FBS (Gibco). Cells are washed, counted, and resuspended in pure IMDMbefore injection. A total of 3×10⁶ CFSE-labeled CD8⁺ OTI cells areinjected i.v. into the tail vein of recipient CD45.1⁺ mice. Forshort-term proliferation studies, OVA-comprising erythroid cells areinjected 24 h following adoptive transfer. Splenocytes are harvested 5 dfollowing antigen administration and stained for analysis by flowcytometry.

Example 7 OVA Antigen Challenge Model in a Mouse

A total of 3×10⁵ CFSE-labeled OTI CD8⁺ T cells are injected into CD45.1⁺recipient mice as described above. At 1 and 6 d following adoptivetransfer, mice are i.v. administered 100 μL of OVA-comprising erythroidcells into the tail vein. At 15 d following adoptive transfer, mice arechallenged with 5 μg of OVA and 25 ng of ultrapure E. coli LPS(InvivoGen) in 25 μL of saline injected intradermally into each rear legpad (Hock method: total dose of 10 μg of OVA and 50 ng of LPS).Quantification of antigen specific B and T cells and serum antibodytiter is described below.

Example 8 Quantification of Antigen Specific B and T Cells in a Mouse

Mice are killed 4 d following OVA challenge, described above, and spleenand draining lymph node cells are isolated for restimulation. For flowcytometry analysis of intracellular cytokines, cells are restimulated inthe presence of 1 mg/mL OVA or 1 μg/mL SIINFEKL peptide (Genscript) for3 h. Brefeldin-A (5 μg/mL; Sigma) is added, and restimulation is resumedfor an additional 3 h before staining and flow cytometry analysis. ForELISA measurements of secreted factors, cells are restimulated in thepresence of 100 μg/mL OVA or 1 μg/mL SIINFEKL peptide for 4 d. Cells arespun, and the media are collected for ELISA analysis using IFN-γ andIL-10 Ready-Set-Go kits (eBioscience) as per the manufacturer'sinstructions.

Example 9 Quantification of Circulating Antibody Titer

OVA-specific serum IgG is detected by incubating mouse serum at varyingdilutions on OVA-coated plates, followed by a final incubation with goatanti-mouse IgG-HRP (Southern Biotech).

Example 10 Extracellular SpyTag-SpyCatcher Tolerance Induction

An expression cassette containing the coding sequence of Kell and SpyTagis synthesized and inserted in the lentiviral vector EF1. A populationof CD34+ cells is transformed with the vector. The expression ofKell-SpyTag fusion protein is quantified by FACS. Cells that expressKell-SpyTag extracellularly are then placed in a solution of Ara h(1-6)peptides fused to the SpyCatcher sequence and a cMyc tag. Followingincubation, the cells are sorted using FACs to quantify covalentisopeptide conjugation of Kell-SpyTag to SpyCatcher-ArahX-cMyc. Thecells are also hypotonically lysed and the presence ofKell-SpyTag-SpyCatcher-ArahX-cMyc is quantified by Western blot.

Example 11 Gene Assembly

DNA encoding the following genes—glycophorin A (Uniprot ID P02724), Kell(Uniprot ID P23276), antibody scFv against hepatitis B surface antigen(Bose et al. 2003 Mol Immunol 40(9):617, GenBank ID AJ549501.1),adenosine deaminase (Uniprot ID P00813), phenylalanine hydroxylase fromChromobacterium violaceum (GenBank ID AF146711.1), complement receptor 1(Uniprot ID P17927), CD46 (GenBank: BAA12224.1), CD55 (Uniprot IDP08174), CD59 (Uniprot ID P13987), green fluorescent protein (Uniprot IDP42212), thymidine phosphorylase (Uniprot ID P19971), glucocerebrosidase(Uniprot ID P04062), beta2 glycoprotein 1 (Uniprot ID P02749),phospholipase a2 receptor (Uniprot ID Q13018), collagen alpha-3(IV)(Uniprot ID Q01955), serum amyloid P (Uniprot ID P02743), lipoproteinlipase (Uniprot ID P06858), asparaginase (Uniprot ID P00805), factor IX(Uniprot ID F2RM35), ADAMTS13 (Uniprot ID Q76LX8)—were purchased as cDNAfrom Dharmacon (GE Life Sciences) or synthesized de novo by DNA2.0 andGenscript.

1. Single Gene Cloning (CR1)

Genes were assembled into expression vectors by standard molecularbiology methods known in the art. The gene for complement receptor 1(CR1) was synthesized by a commercial vendor (DNA2.0) and supplied in astandard cloning vector (pJ series). The gene was amplified out of thepJ vector by polymerase chain reaction (PCR) using oligos withnon-homologous terminal sequences to prepare for insertion into themammalian expression vector (System Biosciences, pM series): theupstream oligo consisted of 25 nt homologous to the upstream pMinsertion site and 25 nt homologous to the start of CR1; the downstreamoligo consisted of 25 nt homologous to the downstream pM insertion siteand 25 nt homologous to the end of CR1. The amplified product waspurified by gel electrophoresis (Qiagen). The pM vector was linearizedby PCR with tail-to-tail oligos homologous to the upstream anddownstream insertion sites and purified by PCR purification (Qiagen).The CR1 amplicon was ligated into the linearized pM vector by Gibsonassembly, described in detail in Gibson 2011, Methods Enzymology Vol498, p. 394. Sequences were confirmed by Sanger sequencing.

2. Fusion of Two Genes (Membrane Kell-scFv)

The gene for Kell was purchased as cDNA and supplied in a standardcloning vector (pJ series). The gene for an antibody scFv specific tohepatitis B surface antigen (scFv, described in Bose 2003, MolecularImmunology 40:617) was synthesized by a commercial vendor (DNA2.0) andsupplied in a standard cloning vector (pJ series). The genes wasamplified out of the pJ vectors by polymerase chain reaction (PCR) usingoligos with non-homologous terminal sequences to prepare for insertioninto the mammalian expression vector (System Biosciences, pM series).Kell was amplified with an upstream oligo consisting of 25 nt homologousto the upstream pM insertion site and 25 nt homologous to the 5′terminus of Kell, and a downstream oligo consisting of 25 nt homologousto the 5′ terminus of scFv and 25 nt homologous to the 3′ terminus ofKell. scFv was amplified with an upstream oligo consisting of 25 nthomologous to the 3′ terminus of Kell insertion site and 25 nthomologous to the 5′ terminus of scFv, and a downstream oligo consistingof 25 nt homologous to the downstream pM insertion site and 25 nthomologous to the 3′ terminus of scFv. The amplified products werepurified by gel electrophoresis (Qiagen). The pM vector was linearizedby PCR with tail-to-tail oligos homologous to the upstream anddownstream insertion sites and purified by PCR purification (Qiagen).The Kell and scFv amplicons were ligated into the linearized pM vectorby one-pot Gibson assembly, described in detail in Gibson 2011, MethodsEnzymology Vol 498, p. 394. Sequences were confirmed by Sangersequencing.

3. Linker-Assembly Between Genes (Kell-scfv)

The gene for Kell was purchased as cDNA and supplied in a standardcloning vector (pJ series). The gene for an antibody scFv specific tohepatitis B surface antigen (scFv, described in Bose 2003, MolecularImmunology 40:617) was synthesized by a commercial vendor (DNA2.0) andsupplied in a standard cloning vector (pJ series). The genes wasamplified out of the pJ vectors by polymerase chain reaction (PCR) usingoligos with non-homologous terminal sequences to prepare for insertioninto the mammalian expression vector (System Biosciences, pM series).Kell was amplified with an upstream oligo consisting of 25 nt homologousto the upstream pM insertion site and 25 nt homologous to the 5′terminus of Kell; and a downstream oligo consisting of 25 nt homologousto the 5′ terminus of scFv, 24 nt encoding a (GlyGlyGlySer)x2 spacer,and 25 nt homologous to the 3′ terminus of Kell. scFv was amplified withan upstream oligo consisting of 25 nt homologous to the 3′ terminus ofKell insertion site, 24 nt encoding a (GlyGlyGlySer)x2 spacer, and 25 nthomologous to the 5′ terminus of scFv; and a downstream oligo consistingof 25 nt homologous to the downstream pM insertion site and 25 nthomologous to the 3′ terminus of scFv. The amplified products werepurified by gel electrophoresis (Qiagen). The pM vector was linearizedby PCR with tail-to-tail oligos homologous to the upstream anddownstream insertion sites and purified by PCR purification (Qiagen).The Kell and scFv amplicons were ligated into the linearized pM vectorby one-pot Gibson assembly, described in detail in Gibson 2011, MethodsEnzymology Vol 498, p. 394. Sequences were confirmed by Sangersequencing.

4. Epitope Tag Attachment (Kell-scFv)

The gene for Kell was purchased as cDNA and supplied in a standardcloning vector (pJ series). The gene for an antibody scFv specific tohepatitis B surface antigen (scFv, described in Bose 2003, MolecularImmunology 40:617) was synthesized by a commercial vendor (DNA2.0) andsupplied in a standard cloning vector (pJ series). The genes wasamplified out of the pJ vectors by polymerase chain reaction (PCR) usingoligos with non-homologous terminal sequences to prepare for insertioninto the mammalian expression vector (System Biosciences, pM series).Kell was amplified with an upstream oligo consisting of 25 nt homologousto the upstream pM insertion site and 25 nt homologous to the 5′terminus of Kell; and a downstream oligo consisting of 25 nt homologousto the 5′ terminus of scFv, 24 nt encoding a (GlyGlyGlySer)x2 spacer,and 25 nt homologous to the 3′ terminus of Kell. scFv was amplified withan upstream oligo consisting of 25 nt homologous to the 3′ terminus ofKell insertion site, 24 nt encoding a (GlyGlyGlySer)x2 spacer, and 25 nthomologous to the 5′ terminus of scFv; and a downstream oligo consistingof 25 nt homologous to the downstream pM insertion site, the 27 ntsequence tacccctatgacgtgcccgactatgcc (Seq. ID No. 8) encoding an HAepitope tag, and 25 nt homologous to the 3′ terminus of scFv. Theamplified products were purified by gel electrophoresis (Qiagen). The pMvector was linearized by PCR with tail-to-tail oligos homologous to theupstream and downstream insertion sites. The downstream primeradditionally contained the 27 nt sequence tacccctatgacgtgcccgactatgcc(Seq. ID No. 8) encoding an HA epitope tag. The linearized vector waspurified by PCR purification (Qiagen). The Kell and scFv amplicons wereligated into the linearized pM vector by one-pot Gibson assembly,described in detail in Gibson 2011, Methods Enzymology Vol 498, p. 394.Sequences were confirmed by Sanger sequencing.

5. Fusion of Two Genes (Reporter Assembly) (GPA-HA)

The genes for complement receptor 1 (CR1) and green fluorescent protein(GFP) were synthesized by a commercial vendor (DNA2.0) and supplied instandard cloning vectors (pJ series). The CR1 gene was amplified out ofthe pJ vector by polymerase chain reaction (PCR) using oligos withnon-homologous terminal sequences to prepare for insertion into themammalian expression vector (System Biosciences, pM series): theupstream oligo consisted of 25 nt homologous to the upstream pMinsertion site and 25 nt homologous to the start of CR1; the downstreamoligo consisted of 54 nt homologous to the viral-derived T2A sequencegagggcagaggaagtcttctaacatgcggtgacgtggaggsgsstcccggccct (Seq. ID No. 7).The GFP gene was amplified out of the pJ vector by polymerase chainreaction (PCR) using oligos with non-homologous terminal sequences toprepare for insertion into the mammalian expression vector (SystemBiosciences, pM series): the upstream oligo consisted of 54 nthomologous to the viral-derived T2A sequencegagggcagaggaagtcttctaacatgcggtgacgtggaggsgsstcccggccct (Seq. ID No. 7)and 25 nt homologous to the start of GFP; the downstream oligo consistedof 25 nt homologous to the downstream pM insertion site and 25 nthomologous to the end of GFP. The amplified products were purified bygel electrophoresis (Qiagen). The pM vector was linearized by PCR withtail-to-tail oligos homologous to the upstream and downstream insertionsites and purified by PCR purification (Qiagen). The CR1 and GFPamplicons were ligated together and into the linearized pM vector byGibson assembly, described in detail in Gibson 2011, Methods EnzymologyVol 498, p. 394. Sequences were confirmed by Sanger sequencing.

Example 12 mRNA Assembly

A gene of interest is cloned into the multiple cloning site of the pSP64vector (Promega) using standard molecular biology methods. The vector isdigested with EcoRI (NEB) to generate a linearized dsDNA vectorcontaining the SP6 promoter, gene of interest, and 30 nucleotide longpoly-A tail. mRNA is synthesized by reaction with SP6 RNA polymerase(Promega) according to manufacturer's instructions, includingrecommended concentrations of 5′ cap analog (ARCA) to synthesize cappedmRNA transcript. The reaction mixture is then treated with DNAse todigest the template vector (Riboprobe from Promega) and the mRNA ispurified using the EZNA MicroElute RNA Clean-Up kit (Omega).

Example 13 Cell Culture

1. Human Red Blood Cells (RBCs)

CD34 cells are isolated from peripheral blood by supermagnetic microbeadselection by the use of Mini-MACS columns (Miltenyi Biotec; 94%+/−3%purity). The cells are cultured in erythroid differentiation medium(EDM) on the basis of IMDM supplemented with stabilized glutamine, 330μg/mL holo-human transferrin, 10 μg/mL recombinant human insulin, 2IU/mL heparin, and 5% solvent/detergent virus-inactivated plasma. Theexpansion procedure comprises 3 steps. In the first step (day 0 to day7), 10{circumflex over ( )}4/mL CD34+ cells are cultured in EDM in thepresence of 1 μM hydrocortisone, 100 ng/mL SCF, 5 ng/mL IL-3, and 3IU/mL EPO. On day 4, 1 volume of cell culture is diluted in 4 volumes offresh medium containing SCF, IL-3, EPO, and hydrocortisone. In thesecond step (day 7 to day 11), the cells are resuspended at10{circumflex over ( )}5/mL in EDM supplemented with SCF and EPO. In thethird step (day 11 to day 18), the cells are cultured in EDMsupplemented with EPO alone. Cell counts are adjusted to7.5×10{circumflex over ( )}5 to 1×10{circumflex over ( )}6 and5-10×10{circumflex over ( )}6 cells/mL on days 11 and 15, respectively.Beyond day 18, the culture medium containing EPO is renewed twice aweek. The cultures are maintained at 37° C. in 5% CO2 in air.

2. Mouse Red Blood Cells

Methods of culturing mouse erythroid cells from mouse fetal livererythroid progenitors are known in the art, see e.g., Shi et al. 2014,PNAS 2014 111(28):10131.

Mouse erythroid progenitors are isolated from fetal livers. Fetal liversare purchased from Charles River Labs. Livers are put in 1 ml PBS onice. Pipette up and down to get a single-cell suspension solution andpass by a 70 um strainer (BD Falcon 35-2235). Rinse the mesh with 1 mlPBS. Combine the flow through (1 ml per embryo). Pellet the cells at 1.5k RPM for 5 min, re-suspend with red cell lysis buffer (AmmoniumChloride Solution from Stemcell), and incubate on ice for 10 mins.Pellet the cells at 1.5 k RPM for 5 min, remove the lysis buffer, andre-suspend with 10 ml PBS-2% FBS. Add chromPure Rat IgG (JacksonImmunoResearch, #012-000-003) at 50 ul/mouse and incubate at 4 C for 5min. Add Biotinylated anti-mouse TER119 (BD Pharmingen, #553672) at (at1 ul/1*10{circumflex over ( )}6 cells) and incubate at 4 C for 15 min.Add Ms Lineage Panel (Fisher Scientific (Thermo Fisher Scientific)#BDB559971) to the cells at (2 ul/1*10{circumflex over ( )}6 cells) andincubate at 4 C for 15 min. Washing once with 10× volume of PBS/and Spinthe cells with 1.5 k RPM for 5 min at 4 degree. Add StreptavidinParticles Plus—DM (magnetic beads) (BD Pharmigen, #557812) (5ul/1*10{circumflex over ( )}6 cells) and incubate at 4 C for 30 min.Prepare 2-4 FACS tubes on a magnetic holder. Aliquot 2 ml cells intoeach tube (4 ml in total), and carefully take the cells out of the tubeand put into the other tube on the other side avoiding the disruption ofthe magnetic stick beads. Repeat the same procedure and take the Ter119negative and linkage negative cells to a new tube. Concentrate thecells, and resuspend the cells with 50-100 ul PBS (2% FBS).

Purified erythroid progenitors are cultured in differentiation mediumcomprising (for 40 mL):IMDM:29 ml, FBS (Stem Cell): 6 ml (Final 15%),10% BSA in IMDM (Stem Cell): 4 ml (Final 1%), 10 mg/ml Holo-transferrin:2000 ul (Final: 500 ug/ml), 100*L-Glutamine: 400 ul, 100*penicillinstreptomycin: 400 ul, 10 U/ul Epo: 2 ul (Final: 0.5 U/ml), 10 mg/mlInsulin: 40 ul (Final: 10 ug/ml). Culture 2*10{circumflex over ( )}5cells/ml in the differentiation medium in 24 wells plate at 37 C. Aftera total culture of 44-48 hours, analyses are performed, for example byflow cytometry as performed herein. Enucleated red blood cells are gatedout using (Hoechst stain) for differentiation profile analysis. Asuccessful culture will yield 16 fold increase.

3. Platelets

Donated CD34+ cells are acquired from the Fred Hutchinson CancerResearch Center. The CD34+ enriched cells are plated in a serum-freemedium at 2-4×10{circumflex over ( )}4 cells/mL and medium refreshmentis done on day 4 by adding an equal volume of media. On day 6, cells arecounted and analyzed: 1.5×10{circumflex over ( )}5 cells are washed andplaced in 1 mL of the same medium supplemented with a cytokine cocktailconsisting of TPO 30 ng/mL, SCF 1 ng/mL, interleukin (IL)-6 7.5 ng/mLand IL-9 13.5 ng/mL] to induce megakaryocyte differentiation. At day 10,½-¼ of the suspension culture is replaced with fresh medium. Allcytokines are purchased from Peprotech. The cultures are incubated in ahumidified atmosphere (10% CO2) at 39° C. for the first 6 days ofculture and 37° C. for the last 8 days. Viable nucleated cells arecounted with a hemocytometer (0.4% trypan blue; Invitrogen, Burlington,ON, Canada).

Clonogenic progenitor cells (CPC) are assayed using MethoCult H4436 formyeloid CPC, and MegaCult-C for colony-forming unit-megakaryocyte(CFU-Mk), according to manufacturer's instructions (StemCellTechnologies, Vancouver, BC, Canada). To assess differentiation, cellsare stained with antibodies against CD61m CD42b, CD41, CD61, and CD49bby flow cytometry using a FACS-Calibur (Becton Dickinson). For cellcycle analysis, cells are rinsed with phosphate-buffered saline (PBS),fixed with formaldehyde 2% (Sigma, St Louis, Mo., USA) for 5 min andpermeabilized with 0.1% of Triton X-100 (Bio-Rad, Hercules, Calif.,USA). Cells are then marked with mAb-Ki-67-FITC (BD Bioscience, SanJose, Calif., USA), washed and resuspended in 0.5 mL PBS-1% fetal bovineserum (FBS)-0.01% azide 7-amino-actinomycin D (7-AAD) following themanufacturer's instructions (BD Biosciences).

Example 14 Cell Isolation

1. Primary RBCs

Whole blood is collected using aseptic techniques in tubes containinglow molecular weight heparin, dalteparin sodium (9 units/mL blood).Blood is centrifuged at 5000×g for 5 minutes and after removal of plasmaand buffy coat (both can be retained for later use), the erythrocytesare washed twice in cold (4 C) phosphate buffered saline (PBS) withcentrifugation. The resultant red blood cell population is stored at 4 Cin CPDA-1 anticoagulant or a glycerol solution for long-termpreservation.

2. Primary Platelets

Whole blood (40 ml) is collected in 3.8% sodium citrate (1:9 citrate toblood vol/vol) from healthy individuals under an appropriate IRBprotocol. Blood is centrifuged at 200 g for 15 minutes to isolateplatelet-rich plasma (PRP). Platelets are then washed in modifiedTyrode's buffer (containing 138 mM NaCl, 5.5 mM dextrose, 12 mM NaHCO3,0.8 mM CaCl2, 0.4 mM MgCl2, 2.9 mM KCl2, 0.36 mM Na2HPO4 and 20 mMHepes, pH 7.4) in presence of 1 μM prostaglandin 12, and resuspended inthe same buffer.

Example 15 Irradiation of Primary or Cultured Cells

Irradiation of a population of exogenous antigen-expressing EHCs can beperformed to ensure that they are incapable of replication. Suchprotocols are similar to those known in the art for irradiating cells,e.g., primary red blood cells. Briefly, one unit (350 ml) of whole bloodis taken and divided into two aliquots of 175 ml each, 10 such units arethus divided into 20 aliquots. One aliquot (175 ml) from each unit ofblood is subjected to gamma irradiation of 25 Gy, and not exceeding 50Gy, by a self-contained gamma cell irradiator (GammaCell 1000,Theratronics). The blood is then stored at 4 C under conventional bloodbanking conditions. Sampling is done from these 10 irradiated and 10non-irradiated blood bags on days 0, 7, 14, and 21 with the help ofsampling site coupler (Fenwal, USA). Tests for cell proliferation areconducted, including a thymidine incorporation assay to quantify anymitotic potential. Supernatant is assayed for free hemoglobin byabsorbance spectroscopy, and for free lactate dehydrogenase bycolorimetric assay (Pierce) to evaluate levels of cell lysis.

Example 16 Enucleation of Erythroid Cells

Erythroid cells are grown to semiconfluence (1 to 4×10{circumflex over( )}4 cells per cm2) on 12-mm diameter coverslips coated with collagenin IMDM medium supplemented with 100 units/mi of penicillin and 100units/ml of streptomycin. The collagen is necessary to prevent all thecells from falling off the coverslip during centrifugation. Cells aregrown to monolayers (5×104 cells per cm2) on coverslips either in thesame medium or in Dulbecco's modified Eagle's medium with 10% calfserum. It is not necessary to coat the cell coverslips with collagen. Inorder to enucleate the cells, the coverslips are inverted (cell sidedown) and placed into the bottom of 15-ml Corex centrifuge tubescontaining 2-5 ml of medium with 10 g of cytochalasin B per ml. Thecentrifuge tubes with the coverslips are placed immediately into aSorvall RC-2 centrifuge that has been warmed to 37 C by spinning the (SS34) rotor with the head in place for about 1 hr at 10,000 rpm (with thetemperature regulator set at 37-39°). The length of time and speed ofcentrifugation are crucial factors for successful enucleation. Cells arespun at 9000 rpm for 1 hr at 37±20 and cells are spun at 6500 rpm for 50mM at 37±−20. After centrifugation, the coverslips are removed from thecentrifuge and placed cell side up into 35-mm (Falcon) tissue culturedishes (Biolquest) containing 3 ml of medium without cytochalasin B.Within 30-60 min at 370, the cells are morphologically normal and 90-99%lacked nuclei. Enucleated cells are removed from the coverslips bytreatment with trypsin-EDTA (Grand Island Biological Co.) and the cellsare suspended in normal medium. The enucleated cells are then replatedin small drops on 22-mm2 coverslips kept in 35-mm tissue culture dishesand placed in an incubator. At time intervals after replating, thecoverslips are mounted on slides (12) and observations on the enucleatesare made with Zeiss phase contrast, polarized light, and Nomarskioptics.

Example 17 Contacting of Cells

1. Nucleic Acid—Transfection

The nucleic acid of interest is scaled up to provide approximately 5 ugnucleic acid per 10{circumflex over ( )}5 EHCs to be loaded, e.g., acell, such as an erythroid cell, a platelet, or a hematopoieticprecursor cell. The nucleic acid is diluted in Opti-MEM Medium (LifeTechnologies) at a ratio of 1 ug to 50 uL medium. The diluted nucleic isthen combined with a transfection reagent (Trans-IT for DNA, Trans-ITmRNA for mRNA, Trans-IT siRNA for siRNA, Mirus Bio) at a 1:1 volumeratio and allowed to form complexes for 5 minutes at room temperature.The nucleic acid complex is added to cells for 12-24 hours. Optionally,after this period of time, the media can be exchanged with fresh mediasuch that the transfection reagents are no longer present.

2. Nucleic Acid—Viral Transduction

The gene of interest is cloned into the multiple cloning site oflentivirus vector pCDH with the MSCV promoter sequence from SystemBiosciences.

Lentivirus is produced in 293T cells by transfecting the cells withlipofectamine 5×10{circumflex over ( )}6 293T cells (Lenti-X 293T CellLine, Clontech catalog #632180) are plated in a P10 petri dish the daybefore transfection. Cell confluency should be around 70%. One plate istransfected per construct. 20 μl (10 μg) pPACKH1 (System Biosciences)plasmid mix+2 μg lenti construct+20 μl Plus reagent (LifeTechnologies,Catalog #11514-015) are combined in 400 μl Optimem and incubated 15 minat RT. 30 μl of LF2000 (LifeTechnologies, Catalog #11668-019) is dilutedinto 400 μl Optimem, added dropwise to DNA mix, and incubated for 15 minRT. DNA mix is added to cells (cells are in 9 ml of Optimem). Cells areincubated for 6 hours and then the medium is changed to DMEM/10% FBS.The virus supernatant is collected 48 hours post-transfection bycentrifugation at 1,500 rpm for 5 minutes. The supernatant is collectedand frozen in 1 ml aliquots at −80° C.

Target cells are transduced at day 3-7 of the culture process describedherein. 5×10{circumflex over ( )}5 cultured cells are plated in 500 μLof medium containing 20 μg/mL polybrene in a 24-well plate. For eachvirus, cells are transduced in triplicate wells. Virus supernatant isadded in another 500 μL of medium and the sample is mixed by pipetting.Infection is achieved by spinoculation, spinning the plate at 2000 rpmfor 90 minutes at room temperature. After spinoculation, the cells areincubated at 37 C overnight, and the next day 1 mL of fresh IMDM mediumwith appropriate cytokines is added.

3. Nucleic Acid—Cationic Polymer

An mRNA ecoding the transgene of interest, and including an upstreampromoter sequence and a downstream poly A tail, can be purchased frommultiple commercial vendors (e.g., IDT-DNA, Coralville Iowa). RNAtransfections are carried out using RNAIMax (Invitrogen, Carlsbad,Calif.) or TRANSIT-mRNA (Mims Bio, Madison, Wis.) cationic lipiddelivery vehicles. RNA and reagent are first diluted in Opti-MEM basalmedia (Invitrogen, Carlsbad, Calif.). 100 ng/uL RNA is diluted 5× and 5μL, of RNAIMax per μg of RNA is diluted 10×. The diluted components arepooled and incubated 15 minutes at room temperature before they aredispensed to culture media. For TRANSIT-mRNA transfections, 100 ng/uLRNA is diluted 10× in Opti-MEM and BOOST reagent is added (at aconcentration of 2 μL, per μg of RNA), TRANSIT-mRNA is added (at aconcentration of 2 μL, per μg of RNA), and then the RNA-lipid complexesare delivered to the culture media after a 2-minute incubation at roomtemperature. RNA transfections are performed in Nutristem xenofree hESmedia (STEMGENT®, Cambridge, Mass.) or Opti-MEM plus 2% FBS. Successfulintroduction of the mRNA transcript into host cells can be monitoredusing various known methods, such as a fluorescent label or reporterprotein, such as Green Fluorescent Protein (GFP). Successfultransfection of a modified mRNA can also be determined by measuring theprotein expression level of the target polypeptide by e.g., WesternBlotting or immunocytochemistry. Similar methods may be followed forlarge volume scale-up to multi-liter (5-10,000 L) culture formatfollowing similar RNA-lipid complex ratios.

4. Nucleic Acid—Electroporation

mRNA ecoding the transgene of interest, and including an upstreampromoter sequence and a downstream poly A tail, can be purchased frommultiple commercial vendors (e.g., IDT-DNA, Coralville Iowa).Electroporation parameters are optimized by transfecting erythroidlineage cells with mRNA transcripts and measuring transfectionefficiency by quantitative RT-PCR with primers designed to specificallydetect the exogenous transcripts. For certain cells preparations,discharging a 150 uF capacitor into 2.5×10{circumflex over ( )}6 cellssuspended in 50 μl of Opti-MEM (Invitrogen, Carlsbad, Calif.) in astandard electroporation cuvette with a 2 mm gap is sufficient forrepeated delivery in excess of 10,000 copies of modified mRNAtranscripts per cell, as determined using the standard curve method,while maintaining high viability (>70%). Cell density may vary from1×10{circumflex over ( )}6 cell/50 μl to a density of 2.5×10{circumflexover ( )}6 cells/500 and require from 110V to 145V to transfect cellswith similar efficiencies measured in transcript copies per cell. Largemulti-liter (5-10,000 L) electroporation may be performed similar tolarge volume flow electroporation strategies similar to methodsdescribed with the above described constraints (Li et al., 2002; Geng etal., 2010).

5. Polypeptide—Liposome

Cells, including primary terminally-differentiated cells e.g.,erythrocytes, can be loaded with exogenous protein on their surface andin their cytoplasm. The loading of proteins can be performed usingliposomes.

Lipids (Pro-Ject reagent, Pierce) in organic solvent were dried undernitrogen into a thin film in glass scintillation vial. Approximately 2uL lipids were used per 10{circumflex over ( )}5 cells. Polyclonal mouseIgG (Abcam) was labeled with Dylight-650 (Pierce) per manufacturer'sinstructions. Protein solution at 0.1 mg/mL in PBS was added to thedried lipid mixture. The solution was pipetted several times, incubatedfor 5 minutes at room temperature, then vortexed vigorously to generateencapsulating liposomes. Serum-free medium was added to bring the totalvolume to 500 uL per 10{circumflex over ( )}5 cells. The liposomalmixture was then incubated with the cells for 3-4 hours at 37 C.

FIG. 1 shows the loading of an exogenous protein, in this casefluorescently-labeled IgG, into primary erythrocytes with liposomes. Theloading is measured by flow cytometry. The loading is dose-dependent, as0.06% of cells are fluorescent without liposomes, ˜60% of cells arefluorescent at a low liposome dose, and ˜85% of cells are fluorescent ata high liposome dose. The data in FIG. 1 is strong proof that exogenousproteins can be loaded into erythroid cells with liposomes.

6. Polypeptide—Mechanical Disruption

Cells may be loaded using a microfluidic device containing 1 μm, 2 μm, 3μm, 4 μm, 5 μm, 10 μm wide channels that transiently porate the cells,allowing a payload to enter when the cells are pressured through thesystem.

The silicon-based devices are fabricated at the Massachusetts Instituteof Technology microfabrication facility using photolithography and deepreactive ion etching techniques. In this process, 6″ silicon wafers witha 450-μm thickness are treated with hexamethyldisilazane, spin coatedwith photoresist (OCG934; FujiFilm) for 60 s at 3,000 rpm, exposed to UVlight (EV1; EVG) through a chrome mask with theconstriction channeldesign, and developed in AZ405 (AZ Electronic Materials) solution for100 s. After 20 min of baking at 90° C., the wafer is etched by deepreactive ion etching (SPTS Technologies) to the desired depth (typically15 μm). The process is repeated on the opposite side of the wafer (i.e.,the one not containing the etched channels) using a different mask,which contains the access hole patterns, and using a thicker photoresistAZ9260 (AZ Electronic Materials). Wet oxidation is then used to grow100-200 nm of silicon oxide before the wafer is anodically bonded to aPyrex wafer and diced into individual devices. Before each experiment,devices are visually inspected and mounted onto a holder with inlet andoutlet reservoirs (all designed in-house and produced by Firstcut).These reservoirs interface with the device using Buna-N O-rings(McMaster-Carr) to provide proper sealing. The inlet reservoir isconnected to a home-made pressure regulator system using Teflon tubingto provide the necessary driving force to push material through thedevice. A population of erythroid cells is first suspended in thedesired delivery buffer [growth medium, PBS, or PBS supplemented with 3%FBS and 1% F-68 Pluronics (Sigma)], mixed with the desired deliverymaterial, and placed in the device's inlet reservoir. This reservoir isconnected to a compressed air line controlled by a regulator, and theselected pressure (0-70 psi) is used to drive the fluid through thedevice. Treated cells are then collected from the outlet reservoir.Cells are incubated at room temperature in the delivery solution for5-20 min after treatment to ensure hole closure before being subjectedto any further treatment. To deliver fluorescently labeled phenylalanineammonia hydroxylase (PAH), the experiments are conducted as describedabove such that the delivery buffer contained 0.1-0.3 mg/mL PAH. GFPknockdown is measured as the percentage reduction in a cell population'saverage fluorescence intensity relative to untreated controls.

7. Polypeptide—Surface Conjugation

The cell surface is treated with Traut's reagent (2-iminothiolane HC1,Pierce) to thiolate primary amines Traut's reagent is dissolved in Trisbuffer pH 8 with EDTA to prevent oxidation of sulfhydryls. Approximately1 pmol Traut's reagent is used to treat 10{circumflex over ( )}6 cells.Incubate Traut's reagent with cells for 1 hour at room temperature.Remove excess or unreacted reagent by centrifugation and washing thecells. The number of available sulfhydryl groups can be measured usingEllman's Reagent. In the meantime, treat suitable exogenous antigenpolypeptide with amine-to-sulfhydryl crosslinker, such as SMCC (Pierce)according to manufacturer's instructions. Excess crosslinking reagent isremoved by desalting. The maleimide-functionalized protein is thenincubated with the thiolated cells for several hours. Unreacted proteinis separated from the conjugated cells by centrifugation and washing.

8. Polypeptide—Non-Covalent Surface Attachment

The gene for an antibody scFv against hepatitis B surface antigen (scFv,described in Bose 2003, Molecular Immunology 40:617) is fused to a6-histidine affinity tag and to the gene encoding the polypeptidesequence that binds mouse glycophorin A, HWMVLPWLPGTLDGGSGCRG, in amammalian expression vector (Genlantis). The full fusion protein isproduced by transient transfection of HEK-293T cells using standardmethods and purified on a Ni-NTA affinity resin (Pierce) according tomanufacturer's instructions. The purified fusion protein is incubatedwith mouse erythrocytes at >100 nM concentration to allow for rapidequilibration and binding of the peptide to glycophorin A.

9. Polypeptide—Lipid Insertion into Membrane

Traut's reagent (Thermo Fisher) is used to generate sulfhydryl groups onan amine-containing suitable exogenous antigen polypeptide moleculefollowing manufacturer's protocol. The reaction mixture is incubated for1 h at room temperature (RT) on a shaker and washed through a spindesalting column (Zeba, MWCO 7K, Thermo Scientific) following themanufacturer's instructions to remove the unreacted Traut's reagent. Thegeneration of sulfhydryl groups on the modified polypeptide isquantified using Ellman's Reagent (Pierce) based on the manufacturer'sprotocol.

DSPE-PEG₃₄₀₀-mal (1×10{circumflex over ( )}-3 M in PBS, 4 μL, molarratio lipid:Polypeptide=1:1) (all lipids purchased from Avanti PolarLipids and stored as chloroform solution under argon at −20 C) are addedto the desalted polypeptide solution and incubated at RT on a shaker.After 1 h, the sample solution is filtered using a centrifugal filterdevice (Microcon, Millipore Co.) at 14 000 g for 15 min at 4° C. toremove the small molecules and suspended in 600 μL PBS (1 mg/mLpolypeptide).

200 μL of whole blood is suspended in 1000 μL PBS and spun at 1500 g for30 s, repeated four times. Finally, the RBCs are suspended in 800 μLPBS. The conjugation of RBC/DSPE-PEG-Polypeptide is prepared by mixingthe above RBCs suspensions and various amounts of DSPE-PEG-Polypeptidesolution (1 mg per mL) followed by incubation for 15-30 min at 37° C.The mixture is kept for 5 min at room temperature, then washed threetimes in PBS and resuspended to a final RBC concentration of5×10{circumflex over ( )}8 per mL. An automated cell counter (Countess,Invitrogen) is used to measure the cell concentration.

10. Polypeptide—Hypotonic Loading

A suitable exogenous antigen polypeptide, in this instance mouse IgG,was purchased from Abcam and was added at 0.25 mg/mL to a RBC suspensionin isotonic solution at a hematocrit (Hct) of 70%. The suspension wasdialyzed in 250 mL of a hypotonic solution containg 10 mM sodiumphosphate pH 7.4, 10 mM sodium bicarbonate, and 20 mM glucose, stirredat 15 rpm for 1 hour at 4 C. The cells were then isotonically resealedby adding 1/10 volume of resealing solution comprising 5 mM adenine, 100mM inosine, 100 mM sodium pyruvate, 100 mM sodium phosphate, 100 mMglucose, 12% (w/v) NaCl at pH 7.4. Cells were then incubated at 37 C for30 minutes.

11. Polypeptide—Cell-Penetrating Peptide

The manufacture of protamine-conjugated polypeptide is known in the art,see e.g., Kwon et al. 2009 J Contr Rel 139(3):182. 5 mg/ml of LowMolecular Weight Protamine (LMWP) in 50 mM HEPES buffer (pH 8) is mixedwith the heterobifunctional cross-linker 3-(2-pyridyldithio)propionicacid N-hydroxysuccinimide (SPDP, Sigma-Aldrich) at a 1:10 molar ratio inDMSO and shaken for 1 h at room temperature. The reaction mixture isthen treated with 50 mM dithiothreitol (DTT, Sigma-Aldrich) and thethiolated LMWP is purified by HPLC on a heparin affinity column. Theproduct is collected by ultrafiltration, lyophilized, and stored at −20°C. until further use.

For conjugation, 5 mg/ml suitable exogenous antigen polypeptide is mixedwith SPDP (40 μl of 0.1 M SPDP in ethanol to 1 ml protein solution) inphosphate buffer, and stirred at room temperature for 1 h. UnreactedSPDP is removed by rapid desalting and buffer exchange by FPLC with 0.1M phosphate buffer (pH 7.4). Activated polypeptide is then conjugatedwith a 10-fold molar excess of the above-prepared LMWP-SH for 24 h at 4°C. The LMWP-polypeptide conjugates are isolated by ion-exchangechromatography using a heparin affinity column followed by five roundsof centrifugal filtration (molecular weight cut-off: 5,000 Da). PooledLMWP-polypeptide conjugates are concentrated, and the degree ofconjugation determined by MALDI-TOF mass spectroscopy.

For uptake experiments, fresh sheep erythrocytes (MP Biomedicals, Solon,Ohio) are suspended in Hank's balanced salt solution (HBSS) at a densityof 5×10{circumflex over ( )}8 cells/ml, and are then incubated with a0.5 mg/ml solution of the LMWP-polypeptide conjugates for 30 min at roomtemperature under gentle shaking. RBCs are then washed with HBSS andstored at 2-8 C.

12. Polypeptide—Chemical Permeability

3×10{circumflex over ( )}8 RBCs were preincubated for 30 min withchlorpromazine (Sigma Aldrich) at 200 μM in Ringer's solution.Afterwards, the suitable exogenous antigen polypeptide was added inRinger's solution (1 to 4 μM) to a final volume of 400 μl and incubatedfor 30 min at room temperature under mild agitation. After incubation,cells were washed twice, resuspended in Ringer and collected foranalysis.

13. Polypeptide—Enzymatic Conjugation

Cell surface enzymatic conjugations with sortase are known in the art,see e.g., Shi et al PNAS 2014 111(28):10131. To label the GPA N terminuswith polypeptide, 30 uL of 500 uM S aureus sortase and 1 mM polypeptidewith LPETGG at the C terminus is preincubated in 50 mM Tris pH 7.5, 150mM NaCl, on ice for 15 minutes and added to 5×10{circumflex over ( )}7RBCs in DMEM. The sortase and cell mixture is incubated on ice for 30min with occasional gentle mixing, then spun at 500×g for 2 min at 4 Cto remove buffer/DMEM, then washed three times with 1 mL of ice-coldPBS.

Example 18 Assessment of Polypeptide Presence

1. Fluorescent Transgene

Erythroid cells were cultured as described herein. A transgene encodingglycophorin A with an HA tag on the C-terminus fused to GFP with anintervening viral T2A peptide was constructed by Gibson assembly asdescribed herein. The transgene was introduced into the erythroid cellsby lentiviral transduction as described herein. Two days aftertransduction, cells were collected, washed in PBS buffer, and analyzedon a flow cytometer (Attune, Life Technologies). Transduction efficiencywas assessed as the percentage of GFP-positive cells in the population.

2. Cell Surface Proteins

For cell surface proteins, the level of protein expression can bedetected as early as 2 days after transfection by flow cytometry withantibodies specific for the protein or for a co-expressed epitope tag.Erythroid cells were cultured as described herein. A transgene encodingglycophorin A with an HA tag at the N-terminus was constructed by Gibsonassembly as described herein. The transgene was introduced into theerythroid cells by lentiviral transduction as described herein. Two daysafter transduction, cells were collected, washed in PBS buffer, andstained with 1:50 dilution of mouse anti-HA antibody (Abcam) for 1 hr.Cells were washed and then stained with a 1:100 dilution of alexa488-labeled goat anti-mouse secondary antibody (Life Technologies) for30 minutes on ice. Cells were washed and analyzed on a flow cytometer(Attune, Life Technologies). Transduction efficiency was assessed as thepercentage of alexa 488-positive cells in the population.

3. Intracellular Proteins

For intracellular proteins, the level of protein expression can bedetected as early as 8-12 hours after transfection by Western Blot.Erythroid cells were cultured as described herein. A transgene encodingadenosine deaminase with an HA tag at the C-terminus was constructed byGibson assembly as described herein. The transgene was introduced intothe erythroid cells by lentiviral transduction as described herein. Twodays after transduction, cells were collected, washed in PBS buffer, andlysed in RIPA cell lysis buffer (Pierce). Cell lysate was denatured byboiling in 100 mM DTT, then loaded onto a NuPage SDS-PAGE pre-cast gel.After electrophoresis and transfer to nitrocellulose membrane, proteinbands were developed by staining with 1:5000 dilution of mouse anti-HAantibody (Abcam) followed by 1:5000 dilution of goat anti-mouse HRP(Pierce), and subsequent treatment with HRP substrate (SuperSignal,Pierce). Images were captured using an Amersham imager (GE healthcare).

Example 19 Contacting Cells with Chemical Modifying Agent

To increase antigen-presenting cell phagocytosis and to promote livertargeting, the cell compositions described herein are treated for 30 minwith 0.15 microM of calcium ionophore A23187 (Sigma Aldrich, SaintQuentin Fallavier, France) at 37 C or with 5 mM of BS3 (Fischer BioblockScientific, Illkirch, France) at room temperature (RT). Afterprocessing, the final products are stored at 2-8 C in suitable buffer.

Example 20 Assessment of Expression and Activity

The expression of exogenous proteins in and on cultured cells can beassessed quantitatively by flow cytometry (if the protein is expressedon the surface) or by Western blot (for proteins expressed in thecytoplasm).

1. Quantitative Flow Cytometry

Anti-mouse Fc-binding quantitative flow cytometry beads (Simply CellularCalibration) were purchased from Bangs Labs. Fluorescently labeled mouseantibodies against relevant cell surface receptors—glycophorin A, Ckit,and transferrin receptor—were purchased from BioLegend. Fluorescentlylabeled mouse antibody against the HA epitope tag was purchased fromLife Technologies. Erythroid cells were cultured as described herein. Atransgene encoding glycophorin A with an HA tag at the N-terminus wasconstructed by Gibson assembly as described herein. The transgene wasintroduced into the erythroid cells by lentiviral transduction asdescribed herein. At least two days after transduction, 2×10{circumflexover ( )}5 cells were collected, washed in PBS buffer, and stained with1:100 dilution of one of the above-listed antibodies for 1 hr. Cellswere washed and analyzed on a flow cytometer (Attune, LifeTechnologies). The protocol was repeated for each of the four antibodieslisted above. Quantification was performed according to manufacturer'sinstructions. Briefly, one drop of each of the five bead samples wasincubated with 1:100 dilution of an above-listed antibody. The beadswere incubated for 1 hr, washed in PBS, and analyzed on a flow cytometer(Attune, Life Technologies). The protocol was repeated for each of thefour antibodies listed above. Calibration curves were fit using themanufacturer's provided excel spreadsheets, from which quantification offluorescence intensity for the cell-based signals was derived.

2. Quantitative Western Blot

Erythroid cells were cultured as described herein. A transgene encodingadenosine deaminase with an HA tag at the C-terminus was constructed byGibson assembly as described herein. The transgene was introduced intothe erythroid cells by lentiviral transduction as described herein. Twodays after transduction, cells were collected, washed in PBS buffer, andlysed in RIPA cell lysis buffer (Pierce).

The transgene was introduced into HEK293T cells by transienttransfection using lipofectamine 2000 (Life technologies). Cells werecultured for one week and the supernatant was harvested. Recombinantprotein was purified on an HA affinity column (Pierce) according tomanufacturer's instructions. Protein concentration was assessed byabsorbance at 280 nm.

Western blotting was performed as described herein. In addition to thecell lysate samples, known amounts of the recombinant adenosinedeaminase were run on the same gel. Following image collection, theintensity of the recombinant bands were used to generate a standardcurve to quantify the amount of protein present in the cell samples.

The robust expression of transgenes at high levels has importantimplications for the therapeutic capacity of the final cell population.FIG. 2 quantifies the expression of three surface proteins indicative ofdifferentiation and one exogenous transgene by quantitative flowcytometry, and demonstrates that the transgene is robustly expressed ata high level.

Erythroid cells in culture were collected at seven time points during afour-stage in vitro differentiation process. At the first time point(“Expand D6”) the cells are nucleated hematopoietic precursors. By thefinal time point (“Diff 3 D8”) the cells are predominantly enucleatederythroid cells. GPA (solid triangles), a canonical marker of erythroidcells, starts low in the precursor cells and rapidlyreaches >1×10{circumflex over ( )}6 copies per cell. CKIT (dashedsquares), a receptor for stem cell factor, starts high then decreases to<1×10{circumflex over ( )}4 copies per cell as differentiation ensues.TR (dotted diamonds), necessary for the transport of iron into erythroidcells, increases initially then gradually declines to <1×10{circumflexover ( )}5 copies per cell. The transgene (open circles) is introducedat the end of the second differentiation stage (“Diff 1”) and issteadily expressed at approximately 1×10{circumflex over ( )}5 copiesper cell throughout differentiation. The above data demonstrate thattransgenes are robustly expressed in cultured cells.

The expression of exogenous proteins in and on cultured cells can beassessed by flow cytometry (if the protein is expressed on the surface)as described herein, or by Western blot (for proteins expressed in thecytoplasm) as described herein. In instances where an exogenous gene isin a single-transcript construct that contains a downstream fluorescentreporter protein, the fluorescence of the reporter protein can be usedas a proxy for expression of the upstream gene, and assessed by flowcytometry as described herein.

FIG. 3A-S shows the exogenous expression of surface and cytoplasmicproteins on enucleated cultured erythroid cells. The above dataconclusively demonstrate that multiple protein classes—includingcytoplasmic, surface, intact, fusions to type I membrane proteins,fusions to type II membrane proteins, fusions to GPI-linked membraneproteins, intracellular fusions, overexpressed, and de novoexpressed—can be expressed on multiple cell types including culturedenucleated erythroid cells, cultured nucleated erthyroid precursorcells, and K562 erythroleukemia cells.

FIGS. 3B and 3F demonstrate the simultaneous expression of two exogenousproteins in an enucleated cultured cell.

In FIG. 3B, Erythroid cells were cultured as described herein. Atransgene construct encoding glycophorin A signal sequence, an HAepitope tag, glycophorin A coding sequence, viral T2A cleavable sequenceand GFP was assembled by Gibson assembly as described herein. Thetransgene was introduced into the erythroid cells by lentiviraltransduction as described herein. The cells were cultured to terminaldifferentiation as described herein. Cells were analyzed by flowcytometry as described herein, using a fluorescent anti-HA antibody andGFP fluorescence to detect expression of both transgenes.

In FIG. 3F, Erythroid cells were cultured as described herein. Atransgene construct encoding glycophorin A signal sequence, antibodyscFv specific to hepatitis B surface antigen, HA epitope tag,glycophorin A coding sequence, viral T2A cleavable sequence and GFP wasassembled by Gibson assembly as described herein. The transgene wasintroduced into the erythroid cells by lentiviral transduction asdescribed herein. The cells were cultured to terminal differentiation asdescribed herein. Cells were analyzed by flow cytometry as describedherein, using a fluorescent anti-HA antibody and GFP fluorescence todetect expression of both transgenes.

Example 21 Expression of Protein from mRNA in Platelets

The expression in platelets of exogenous proteins translated fromexogenous transfected mRNA was measured by flow cytometry. In brief,platelet-enriched serum was centrifuged at 190 g for 15 minutes toremove erythrocytes and leukocytes. The supernatant was then spun for anadditional 5 minutes at 2500 g to pellet platelets. Platelets wereresuspended in 5 mL of Tyrode's buffer with 1 uM prostaglandin, washed,and resuspended in 750 uL of Tyrode's buffer with 1 uM prostaglandin.mRNA encoding the gene of interest, in this example GFP, was mixed withlipofectamine at a 1:1 mg/mL ratio. The mixture was incubated for 5minutes, then added to the washed platelet population. The combinationwas incubated for 24 hours at room temperature with slow rocking.Platelet expression of the transgene was assayed by flow cytometrymeasuring GFP fluorescence. Surface proteins can also be assayed by flowcytometry. Cytoplasmic or other intracellularly-expressed proteins canalso be assayed by Western blot.

There is therapeutic relevance to introducing exogenous proteins intoand onto platelets. Since platelets do not possess a nucleus or RNAtranscription machinery, DNA transfection is not a viable means ofinducing exogenous protein expression in platelets. However,mRNAtransfection and translation is a way of introducing exogenous proteinsinto cells. It is thought that platelets contain mRNA translationmachinery, but until now it was not known whether they are able toaccept and translate exogenous mRNA into protein.

FIG. 4 is a collection of flow cytometry plots that demonstrate thetranslation of exogenous mRNA encoding a transgene, in this case GFP, byplatelets. The GFP is detected by fluorescence in the FL1 channel afterexcitation with a 488 nm laser. (4A) Untransfected platelets (1.7%GFP+). (4B) Platelets transfected with 3 ug GFP mRNA (8.6% GFP+). (4C)Platelets transfected with 6.8 ug GFP mRNA (3.3% GFP+).

The data conclusively demonstrate, for the first time, the translationof exogenous mRNA into exogenous protein by platelets.

Example 22 Activity of Enzymes

FIG. 5 demonstrates the activity for enzymes contained on erythroidcells. Biochemical activity of cytoplasmic enzymes was assessed byWestern blot for retention of a protein over the course ofdifferentiation. Biological activity of cytoplasmic enzymes was assessedby in vitro enzymatic activity assay.

FIG. 5 shows the activity of two different intracellular enzymesexpressed in cultured erythroid cells.

1. Adenosine Deaminase.

A transgene encoding adenosine deaminase with an HA tag at theC-terminus was constructed by Gibson assembly as described herein. Thetransgene was introduced into HEK-293T cells by lipofectaminetransfection (Life Technologies) as described herein. Enzymatic activityis assayed using a protocol derived from Helenius 2012, Biochim BiophysActa 1823(10):1967, in which a specific mixture of enzymes convertpurines into uric acid and H2O2 followed by fluorometric detection ofthe generated H2O2. In brief, two days after transfection, cells werecollected, media aspirated, and Krebs Ringer phosphate glucose (KRPG;comprising: 145 mM NaCl, 5.7 mM sodium phosphate, 4.86 mM KCl, 0.54 mMCaCl2, 1.22 mM MgSO4, and 5.5 mM glucose; pH 7.35) added to the cells at2×10{circumflex over ( )}5 cells/mL. Adenosine was added at 50 uM. Afterreaction for 6 hours, supernatant was collected and heat inactivated for5 minutes at 60 C. Aliquots of supernatant were transferred to wells ina white 96-well microplate containing 0.25 U/ml bacterial purinenucleoside phosphorylase (PNP) and 0.15 U/ml microbial xanthine oxidase(XO), both from Sigma. After 20 min incubation at RT, 30 μl ofH2O2-detecting mixture containing HRP (final concentration 1 U/ml,Sigma) and Amplex Red reagent (60 μM, Invitrogen, Molecular Probes) wasadded to the microwells, followed by measurement of the fluorescenceintensity at the emission and excitation wavelengths of 545 and 590 nm,respectively (Tecan Infinite M200).

2. Phenylalanine Hydroxylase

Erythroid cells were cultured as described herein. A transgene encodingphenylalanine hydroxylase with an HA tag at the C-terminus wasconstructed by Gibson assembly as described herein. The transgene wasintroduced into the erythroid cells by lentiviral transduction asdescribed herein. Two days after transduction, cells were collected,washed in PBS buffer, and lysed in RIPA cell lysis buffer (Pierce). Celllysates (64 ug total protein) were added to 1 mL reaction buffercontaining 100 mM Tris-HCl, pH 7.5, 4 mM DTT, 4 mM Phenylalanine, 33 μgcatalase, and 0.4 mM DMPH4 (all from Sigma). Reactions were runovernight at 37 C. After incubation, samples were de-proteinized bycentrifugal filtration in an Amicon Ultra-4 Centrifugal Filter 10 KD(Millipore UFC801024) spinning at 3700 rpm for 10 min. Samples werecollected and assayed for tyrosine concentration by absorbance at 540nm.

Both of these exogenous proteins were retained through the end ofterminal differentiation, a non-trivial feat given that it is well-knownin the field that erythroid cells undergo a rigorous program ofelimination of proteins unnecessary for basic function (Liu J et al.(2010) Blood 115(10):2021-2027, Lodish H F et al. (1975) DevelopmentalBiology 47(1):59). In FIG. 5A, the exogenously over-expressed proteinadenosine deaminase is detected by anti-HA Western blot at various timepoints over the course of differentiation, from nucleated precursorcells (“Diff I D5”) through to enucleated erythroid cells (“Diff IIID8”). In FIG. 5C, the exogenously expressed microbial proteinphenylalanine hydroxylase is detected by anti-HA Western blot at varioustime points over the course of differentiation, from nucleated precursorcells (“Diff I D5”) through to enucleated erythroid cells (“Diff IIID8”).

Additionally, both of these enzymes maintained their ability toenzymatically convert substrate into product. FIG. 5B shows theenzymatic conversion of adenosine to inosine by intact adenosinedeaminase-expressing 293T cells. FIG. 5D shows the enzymatic conversionof phenylalanine to tyrosine by lysates of cultured phenylalaninehydroxylase-expressing enucleated erythroid cells.

These data conclusively demonstrate that exogenous enzymes are retainedon erythroid cells throughout the culture process and that they areenzymatically active in erythroid cells, which has profound therapeuticimplications.

Example 23 Activity of CR1

FIG. 6 shows both biochemical and biological activity for complementreceptor 1 (CR1) over-expressed on the surface of cultured erythroidcells. Biochemical activity of CR1 was assessed by flow cytometry forbinding to an immune complex. Biological activity of CR1 was assessed bytransfer of immune complexes to macrophages in a co-culture assay.

1. Immune Complex Binding of CR1-Expressing Cells.

Erythroid cells were cultured as described herein. A transgene constructencoding complement receptor 1 (CR1) was constructed by Gibson assemblyas described herein. The transgene was introduced into the erythroidcells by lentiviral transduction as described herein. Transgeneexpression levels were assessed by flow cytometry as described hereinusing an anti-CR1 antibody (Abcam). The cells were cultured to terminaldifferentiation as described herein.

Dylight 650-labeled bovine serum albumin (BSA-650) was incubated withpolyclonal rabbit anti-BSA (Abcam) in an excess of antibody for 30minutes at room temp. The complexes were then mixed with human serum ata 1:1 volume ratio for 30 minutes at 37 C. Control complexes were eithernot mixed with human serum or mixed with heat-inactivated human serum.

Complexes were incubated with the CR1-expressing cells for 30 minutes at37 C. Cells were washed and analyzed by flow cytometry for capture ofimmune complexes by detecting Dylight 650 fluorescence.

2. Immune Complex Transfer to Macrophages

Cultured U937 monocytes were activated by incubation with 100 nM phorbolmyristate acetate (PMA) for 24 hours at 37 C. Cells coated with immunecomplexes (see above) were incubated with activated U937 macrophages for30 minutes at 37 C. The co-culture was analyzed by flow cytometry.Macrophages were identified by FSC/SSC gating. Presence of immunecomplex on macrophages was analyzed by detecting Dylight 650fluorescence in the macrophage population.

FIG. 6 shows the biochemical and biological activity of complementreceptor 1 (CR1) exogenously over-expressed on cultured erythroid cells.

FIG. 6A shows the biochemical activity of CR1, defined as the capture ofimmune complexes in vitro. The black histogram shows the capture ofBSA-based immune complexes by CR1 over-expressed on cultured erythroidcells. The shaded histogram shows the minimal background binding tocomplexes of BSA and IgG that lack human complement, demonstrating thatthe binding event is CR1-mediated.

FIG. 6B shows the biological activity of CR1, defined as the transfer ofcaptured immune complexes from cultured erythroid cells to macrophages.This is a standard assay in the field, see: Repik et al. 2005 Clin ExpImmunol. 140:230; Li et al. 2010 Infection Immunity 78(7):3129. Transferis assessed by flow cytometry and measured as the intensity of labeledimmune complex-derived fluorescence on macrophages. In this assay,macrophages that are incubated with no immune complexes (black bars) donot become fluorescent. Macrophages that are incubated with complexes ofBSA and IgG that lack complement (and therefore do not bind CR1) take uponly a small amount of immune complex (solid gray bars), independent ofthe presence of cultured CR1-overexpressing erythroid cells. This uptakeis likely due to Fc-gamma receptors on the U937 cells interacting withthe Fc regions of the IgG molecules. Macrophages that are incubated withimmune complexes (BSA+IgG+complement) in the absence ofCR1-overexpressing cells (hashed bar, left) take up the same amount ofimmune complex as in the absence of complement, likely by the sameFc-gamma mediated method. However, the macrophages that are incubatedwith immune complexes in the presence of CR1-overexpressing cells(hashed bar, right) take up nearly double the number of immune complexesas measured by fluorescence.

These data conclusively demonstrate that CR1 overexpression on culturederythroid cells enables the capture of immune complexes on saiderythroid cells, facilitates the transfer of immune complexes fromerythoroid cells to macrophages, and significantly increases the rateand number of immune complexes taken up by macrophages.

Example 24 Activity of scFv

FIG. 7 shows the biochemical and biological activity of antibody scFvexogenously expressed on the surface of cultured erythroid cells as afusion to the transmembrane protein GPA.

Erythroid cells were cultured as described herein. A transgene constructencoding the leader sequence of glycophorin A, an antibody scFv specificto hepatitis B surface antigen (scFv, described in Bose 2003, MolecularImmunology 40:617), an HA epitope tag, a [Gly-3-Ser]2 flexible linker,and the body of glycophorin A was assembled by Gibson assembly asdescribed herein. The transgene was introduced into the erythroid cellsby lentiviral transduction as described herein. Transgene expression wasassessed by flow cytometry as described herein using an anti-HA antibody(Abcam). The cells were cultured to terminal differentiation asdescribed herein. Biochemical activity of the antibody scFv was assessedby flow cytometry for binding to the target protein, in this casehepatitis B surface antigen (HBsAg). Recombinant HBsAg protein (Abcam)was labeled with Dylight-650 fluorophore (Pierce). scFv-expressing cellswere incubated with 100 nM labeled protein, washed in PBS, and analyzedfor Dylight 650 fluorescence by flow cytometry as described herein.

Biological activity of the antibody scFv was assessed by in vivo captureof HBsAg detected by flow cytometry. Recombinant HBsAg protein (Abcam)was labeled with Dylight-650 fluorophore (Pierce). scFv-expressing cellswere fluorescently labeled with CFSE (Sigma) Immunocompromised NSG mice(Jackson labs) were injected with ˜400 pmol of the labeled HBsAg intothe tail vein. A few minutes later, the same mice were injected with2×10{circumflex over ( )}7 scFv-expressing cells. Blood was collected bysubmandibular puncture at regular intervals in an EDTA-containing tube.Collected blood cells were washed and analyzed by flow cytometry asdescribed herein. Human cells were identified as those that were CFSEpositive. Capture of HBsAg was detected as Dylight 650 fluorescence onthe human cells.

FIG. 7A-7B show the biochemical activity of antibody scFv, defined asthe binding of its cognate antigen, hepatitis B surface antigen (HBsAg).In FIG. 7A, cells that express (black) or do not express (gray shaded)the antibody scFv are incubated with 450 nM HBsAg and stained withbiotinylated anti-HBsAg antibody and fluorescent streptavidin. Cellsthat express the antibody scFv (˜45% of the cells in this culture) bindto the antigen. In FIG. 7B, cells that express the antibody scFv areincubated with various concentrations of HBsAg and stained as above,showing that the binding event is dose-dependent with an affinity ofapproximately 10 nM.

FIG. 7C-7D show the biological activity of antibody scFv, defined as thecapture of cognate antigen HBsAg while in circulation in a mouse. Inthis experiment, immunocompromised NSG mice were injected with ˜400 pmolfluorescently-labeled HBsAg via the tail vein. Five minutes later,cultured enucleated erythroid cells (7C) or cultured enucleatederythroid cells that expressed exogenous antibody scFv (7D) wereinjected via the tail vein. Prior to injection, all cultured cells werelabeled with CFSE fluorescent dye. Blood was collected 6 hours later,analyzed on a flow cytometer, and gated on CFSE+ human cells. Barecultured cells did not bind to HBsAg (7C), whereas antibodyscFv-expressing cells do bind to HBsAg (7D). Consistently with thebiochemical activity experiment, approximately 45% of the cells in thisculture express antibody-scFv.

These data demonstrate that the antibody scFv is biochemically activewhen expressed on the surface of cultured erythroid cells and that theantibody scFv on the erythroid cell is able to bind its target in vivowhen in circulation. This has profound implications for therapeuticapproaches in which the capture, sequestration, and clearance of asubstance in circulation is desired.

Example 25 Activity—Circulating Clearance

FIG. 8 shows both biochemical and biological activity for surfacemolecule capture agents used for circulating clearance of a target.

Biochemical activity of the capture agents, in this case HA polypeptideand biotin, was assessed by flow cytometry for binding to the targetprotein, in this case anti-HA antibody and anti-biotin antibody.Biological activity of the capture agents was assessed by in vivocapture and clearance of target protein as detected by flow cytometryand plasma protein quantification.

1. Capture of Anti-Biotin Antibody by Chemically-Modified Cells

Eyrthrocytes from a normal human donor were purchased (Research BloodComponents). Cells were labeled with CFSE (Sigma) per manufacturer'sinstructions for 20 minutes at 37 C. Cells were then biotinylated withNHS-biotin (Sigma) per manufacturer's instructions using 0.02 volumes of2 mM stock biotin reagent for 30 minutes at room temperature.Anti-biotin antibody (Abcam) was fluorescently labeled with Dylight 650(Pierce). Labeling efficiency of the cells was assessed by flowcytometry using the labeled anti-biotin antibody and CFSE fluorescenceas detection markers. 250 ug labeled antibody was injected into an NSGmouse (Jackson Labs) intravenously via the tail vein. Four hours later1×10{circumflex over ( )}8 biotinylated cells were injectedintravenously via the tail vein. Blood was collected by submandibularpuncture at regular intervals in an EDTA-containing tube. Collectedblood cells were washed and analyzed by flow cytometry as describedherein. Human cells were identified as those that were CFSE positive.Capture of anti-biotin antibody was detected as Dylight 650 fluorescenceon the human cells. Plasma from the blood draw was analyzed by ELISAusing a biotin-coated microplate (Pierce) per manufacturer'sinstructions to detect the level of antibody in circulation.

2. Capture of Anti-HA Antibody by Transgenic Cultured Cells

Erythroid cells were cultured as described herein. A transgene constructencoding glycophorin A signal sequence, an HA epitope tag, glycophorin Acoding sequence, viral T2A cleavable sequence and GFP was assembled byGibson assembly as described herein. The transgene was introduced intothe erythroid cells by lentiviral transduction as described herein. Thecells were cultured to terminal differentiation as described herein.Cells were analyzed by flow cytometry as described herein, using ananti-HA antibody (Life Technologies) fluorescently labeled with Dylight650 (Pierce) and GFP fluorescence to detect expression of bothtransgenes. 250 ug labeled anti-HA antibody was injected into an NSGmouse (Jackson Labs) intravenously via the tail vein. Four hours later1×10{circumflex over ( )}8 cultured cells were injected intravenouslyvia the tail vein. Blood was collected by submandibular puncture atregular intervals in an EDTA-containing tube. Collected blood cells werewashed and analyzed by flow cytometry as described herein. Human cellswere identified as those that were CFSE positive. Capture of anti-HAantibody was detected as Dylight 650 fluorescence on the human cells.Plasma from the blood draw was analyzed by ELISA using an HApeptide-coated microplate (Pierce) per manufacturer's instructions todetect the level of antibody in circulation.

FIG. 8 shows biochemical and biological activity of (8A-8B) thepolpeptide HA expressed on the surface of cultured erythroid cells as afusion to GPA and of (8C-8D) biotin chemically conjugated to the surfaceof primary erythrocytes. Biochemical activity is defined as the captureof a target protein in vitro. Biological activity is defined as theenhanced clearance of a target protein in vitro.

In FIG. 8A, the HA polypeptide, expressed as a fusion to the N terminusof GPA, captures a mouse anti-HA antibody in vivo. NSG mice wereinjected with fluorescently-labeled mouse anti-HA antibody, followed byinjection of cultured human erythroid cells that either do not (top) ordo (bottom) express HA epitope tag on their surface as a fusion to GPA.Blood was drawn and cells analyzed on the flow cytometer. The x-axismeasures CFSE fluorescence. The y-axis measures anti-HA antibody Dylight650 fluorescence. CFSE-positive cultured human erythrocytes are observedin both samples, but only the cells expressing the HA epitope tag areable to capture circulating anti-HA antibody.

In FIG. 8B, mice were injected with anti-HA antibody then thenoptionally with cultured human erythroid cells that either do not or doexpress HA peptide on their surface as a fusion to GPA. Plasma wascollected at multiple time points and the level of anti-HA antibody inplasma was assessed by ELISA using an HA peptide-coated plate as asubstrate. Mice injected with anti-HA antibody alone (open circles,solid line—this mouse died after 120 minutes of causes unrelated totreatment) or with anti-HA antibody followed by cells that do notexpress HA peptide on their surface (dashed line) have significantantibody in circulation out to 24 hours post injection of cells. Incontrast, mice injected with anti-HA antibody followed by cells thatexpress HA peptide on their surface are depleted of target antibodywithin minutes. This data conclusively demonstrates that the targetantibody is rapidly and specifically cleared from circulation bycultured erythroid cells that express exogenous antigen polypeptide ontheir surface.

In FIG. 8C, the biotin molecule, conjugated to the surface of erythroidcells by amine functionalization chemistry, captures a mouse anti-biotinantibody. In both of these cases capture was assessed by flow cytometry.Cells that are CFSE labeled and biotinylated show up as double positivewhen stained with a fluorescent anti-biotin antibody (lower dot plot),whereas CFSE-labeled cells that are not biotinylated only show up assingle positive (upper dot plot).

In FIG. 8D, mice were injected with anti-biotin antibody then thenoptionally with cultured human erythroid cells that either are not orare conjugated to biotin on their surface. Plasma was collected atmultiple time points and the level of anti-biotin antibody in plasma wasassessed by ELISA using a biotin-coated plate as a substrate. Miceinjected with anti-biotin antibody alone (open circles, solid line) orwith anti-biotin antibody followed by cells that are not conjugated tobiotin on their surface (dashed line) have significant antibody incirculation out to 24 hours post injection of cells. In contrast, miceinjected with anti-biotin antibody followed by cells that are conjugatedto biotin on their surface are depleted of target antibody withinminutes. This data conclusively demonstrates that target antibodies arerapidly and specifically cleared from circulation by cultured erythroidcells that contain exogenous antigen polypeptide on their surface.

Together the data conclusively demonstrate that suitable exogenousantigens on exogenous antigen-expressing EHCs are able to bind theirtarget molecules in vivo and mediate rapid circulating clearance oftarget molecules when in circulation, which has profound therapeuticimplications.

Example 26 Activity of Complement Regulators

The complement regulatory activity of the exogenous antigen-expressingEHCs is assessed by standard CH50 and AH50 assays known in the art (seee.g., Kabat et al. 1961 Exp Immunochem pp. 133-239 and Platts-Mills etal. 1974 J Immunol 113:348).

Briefly, the CH50 assay utilizes sheep erythrocytes (SRBC) as targetcells. Briefly, a suspension containing 1×10{circumflex over ( )}9SRBC/ml is prepared in the GVB(2+) buffer (gelatin/Veronal-bufferedsaline with Ca2+ and Mg2+), pH 7.35. Hemolysin (rabbit anti-sheepantiserum) is titrated to determine the optimal dilution to sensitizeSRBC. Diluted hemolysin (1:800) is mixed with an equal volume of SRBC(1×109 SRBC/ml), and the whole is incubated at 37° C. for 15 minutes.This results in 5×10{circumflex over ( )}8/ml antibody-coatederythrocytes (EA). EA (100 μl) are incubated with 100 μl of five serialtwofold dilutions (1:20, 1:40, 1:80, 1:160, and 1:320) of the normalhuman serum (NHS) or similar dilution of the mixture of NHS and theexogenous antigen-expressing EHC at 37° C. for 1 hour. NHS incubatedwith GVB2+ buffer is used as the control. Background control is obtainedby incubating EA with buffer alone (serum is not added), and total lysis(100% hemolysis) is determined by adding distilled water to EA. Thereaction is stopped using 1.2 ml of ice-cold 0.15 M NaCl, the mixture isspun to pellet the unlysed cells, and the optical density of thesupernatant is determined spectrophotometrically (412 nm). Thepercentage of hemolysis is determined relative to the 100% lysiscontrol. Complement activity is quantitated by determining the serumdilution required to lyse 50% of cells in the assay mixture. The resultsare expressed as the reciprocal of this dilution in CH50 units/ml ofserum.

Briefly, the AH50 assay depends on lysis of unsensitized rabbiterythrocytes (Erab) by human serum by activation of the alternativepathway. Activation of the calcium-dependent classical pathway isprevented by addition of the calcium chelator ethylene glycoltetraacetic acid (EGTA) to the assay buffer, and magnesium, necessaryfor both pathways, is added to the buffer. Briefly, a cell suspension ofrabbit RBC (2×10{circumflex over ( )}8 cell/ml) is prepared in theGVB-Mg2+-EGTA buffer. A serial 1.5-fold dilution (1:4, 1:6, 1:9, 1:13.5,and 1:20.25) of normal human serum (NHS) or similar dilution of themixture of NHS and the exogenous antigen-expressing EHC are prepared inGVB-Mg2+-EGTA buffer, and 100 μl of each serum dilution is added to 50μl of standardized Erab. NHS incubated with GVB-Mg2+-EGTA buffer is usedas the control. The mixture is then incubated at 60 minutes at 37° C. ina shaking water bath to keep cells in suspension, and 1.2 ml of ice-coldNaCl (0.15 M) is used to stop the reaction. The tubes are spun at 1250g, at 4° C., for 10 minutes to pellet the cells, and the optical densityof the supernatant is determined spectrophotometrically (412 nm).Background control has 100 μl GVB-Mg2+-EGTA buffer, and 50 μl Erab anddoes not exceed 10% of the total lysis. In the total lysis control tube100 μl of distilled water is added to 50 μl Erab suspension, and thepercentage of hemolysis is determined relative to 100% lysis control.The results of the assay are calculated and complement activity isquantitated by determining the serum dilution required to lyse 50% ofcells in the assay mixture. The results are expressed as the reciprocalof this dilution in AH50 units/ml of serum.

Example 27 Activity of Platelet-Loaded Thymidine Phosphorylase

A transgene encoding thymidine phosphorylase with an HA tag at theC-terminus is constructed by Gibson assembly as described herein.Platelets are cultured from precursor cells as described herein. Thetransgene is introduced into the cultured platelet precursor cells bylentiviral transduction as described herein. Expression of thymidinephosphorylase within the cultured platelets is assessed by Westernblotting using an anti-HA detection antibody, as described herein.

Thymidine phosphorylase activity is determined in platelet samples byquantifying the rate of conversion of thymidine to thymine. Preliminaryexperiments are conducted to determine the linear metabolite formationkinetics with respect to time and enzyme dilution; the method is shownto be linear for up to 16 min, over a thymine phosphorylase range of4.0-719 nmol/min/ml (corresponding to a sample dilution range of10-9088). Lysates of pre-dialysis samples cultured platelet and controlplatelet samples are prepared by diluting thawed samples 1:710 with 125mM Tris-HCl, pH 7.4. Twenty-five ul of the platelet lysate is then addedto 100 ul sodium phosphate buffer (100 mM, pH 6.5) and 25 ul thymidinestandard (10 mM), mixed and incubated at 37 C for 10 min. The reactionis terminated with 25 ul of 40% TCA. Assay blanks are prepared by addingTCA to the sodium phosphate buffer/thymidine incubation mixture prior toadding the platelet lysate. Samples are centrifuged at 13,400×g for 2min, and the supernatant washed twice with water-saturated diethyl etherwith 2 min on a shaker to extract the TCA. To avoid ether interferingwith HPLC separation, effective removal is achieved by exposing thematrix to the air for 5 min to allow evaporation of the ether. A samplevolume of 10 ul is injected into the HPLC.

Chromatographic separation of substrate and product is achieved usingreversed phase chromatography with isocratic elution using a WatersAlliance HPLC 2795 system. A pre-packed C18column (Spherisorb ODS 125mm×4.6 mm ID, 5 um particle size, Waters) is used as the stationarystage. Analytes are eluted using a mobile phase of ammonium acetate (40mM) with the ion-pairing agent tetrabutyl ammonium hydrogen sulphate (5mM) adjusted to pH 2.70 with HCl, delivered at a flow rate of 1.0ml/min, with a run time of 8 min. UV detection is at 254 nm and 0.1absorbance units full scale. Metabolites are identified by comparingspectra with pure standards.

Example 28 Activity of Platelet-Displayed Goodpasture Antigen

A transgene encoding collagen alpha-3(IV) (COL4A3) NCl domain antigenfused to the N terminus of CD42b (GP1B, genbank AAH27955.1) with anintervening HA tag is constructed by Gibson assembly as describedherein. Platelets are cultured from precursor cells as described herein.The transgene is introduced into the cultured platelet precursor cellsby lentiviral transduction as described herein. Expression of theexogenous antigen on the cultured platelets is assessed by flowcytometry using an anti-HA detection antibody as described herein.

Serum is collected from a patient suffering from Goodpasture's syndrome,and the serum is tested for anti-COL4A3 antibodies by commercial ELISA(MyBioSource COL4A3 ELISA Kit). The binding capacity of theantigen-expressing platelets is assessed by flow cytometry as describedherein, using this anti-COL4A3 serum as the primary detection antibodyand fluorescent anti-human IgG as the secondary detection antibody.

Platelet-facilitated clearance of a circulating antigen in vivo ismodeled in a mouse using the antigen-expressing platelets. NSG mice areinjected with 100 uL of mouse anti-human COL4A3 antibody (CreativeBioMart) fluorescently labeled with Dylight 650 dye. CFSE-labeledcultured platelets (10{circumflex over ( )}8 per mouse) that express theexogenous antigen are then injected via the tail vein. Blood is drawnfrom a submandibular location at 10 min, 30 min, 2 h, 12 h, and 24 h.Blood is centrifuged to collect the platelet-rich fraction, which isthen stained and analyzed by flow cytometry as described herein.Antibody capture by platelets is determined by tracking the CFSE-Dylight650 double positive population.

Example 29 Activity In Vivo (Mouse)

Mouse erythroid cells are cultured as described herein. Erythroidprecursor cells are transduced with a suitable exogenous antigenpolypeptide transgene, e.g., encoding complement receptor 1 (CR1) usinga lentivirus as described herein. Cells are cultured to terminaldifferentiation as described herein. The presence of the exogenousprotein in the cells is assessed by flow cytometry as described herein.The cells are labeled with a fluorescent die, e.g., CFSE (Sigma Aldrich)per manufacturer's instructions to aid in their detection. The cells areinjected into a NZBWF1/J mouse model of lupus, or other appropriatemodel of disease or activity corresponding to the suitable exogenousantigen polypeptide, approximately 1×10{circumflex over ( )}8 cellsinjected via the tail vein. Blood is collected at multiple time pointsby submandibular puncture Immune complex levels in the plasma aredetected by Raji cell assay, see e.g., Theofilopoulos et al. 976, J ClinInvest 57(1):169. Pharmacokinetics of the cultured cells are assessed byflow cytometry as described herein, by tracking the percentage of CFSEfluorescent cells in the drawn blood sample. Mouse overall health isassessed by gross necropsy, including histology of kidney tissue totrack reduction of immune complex deposition and inflammation-mediateddamage.

Example 30 Rapid Screening

Cell lines, e.g., 293T and K562, have shorter expression and culturingcycles (˜1 day) compared to cultured erythroid cells (days-weeks). Thesecell lines can be used to rapidly iterate through a gene libraryencoding suitable exogenous antigen polypeptides to identify theexogenous antigen polypeptide with the highest expression or activity.

A library of suitable exogenous antigen polypeptide transgenes, e.g.,full-length and shorter variants of complement receptor 1 (CR1), areconstructed by polymerase chain reaction and Gibson assembly asdescribed herein. The library of transgenes is transfected into HEK293Tcells in a parallel fashion in a microtiter plate using lipofectamine asdescribed herein and transduced into K562 cells using lentivirus asdescribed herein. The expression of the exogenous antigens is assessedby flow cytometry as described herein after 24-48 hours. The activity ofeach of the exogenous antigens in the library is assessed by capture offluorescent immune complex detected with flow cytometry as describedherein, and by the transfer of fluorescent immune complexes to culturedmonocytes detected with flow cytometry as described herein. Theexogenous antigens from the library that are most functional—e.g., arehighest expressed, capture most immune complexes, or best transferimmune complexes to monocytes—are then individually transduced intoparallel erythroid cell cultures as described herein using lentivirus asdescribed herein. The expression of each exogenous antigen on culturederythroid cells is assessed by flow cytometry as described herein Theactivity of each exogenous antigen on cultured erythroid cells isassessed by capture of fluorescent immune complex detected with flowcytometry as described herein, and by the transfer of fluorescent immunecomplexes to cultured monocytes detected with flow cytometry asdescribed herein.

Example 31 Assessment of Clearance Rate of RBC In Vivo

The clearance rate of erythroid cells was assessed in vivo in animmunocompromised mouse model. NSG mice were treated at day −1 with 100uL of clordonate liposome (Clodrosomes.com) solution to selectivelydeplete macrophages. Cells were labeled with the fluorescent tag CFSEand approximately 1×10{circumflex over ( )}8 cells were injected intoeach mouse via the tail vein. At regular intervals blood was collectedby submandibular puncture and blood cells were collected. Cells wereco-stained with anti-human GPA antibodies and analyzed by flowcytometry. Human erythroid cells were distinguished from mouse erythroidcells by CFSE signal and by human GPA signal.

For therapeutic applications, it is important that cultured erythroidcells and cultured erythroid cells containing exogenous protein eitherintracellularly or on the surface circulate normally in vivo. This isshown in FIG. 9 using a standard immunocompromised mouse model. In FIG.9A, blood collected from an injected mouse is analyzed on the flowcytometer. Cultured human erythroid cells are identified in the topright quadrant of the plot, double-positive for CFSE and human-GPA. InFIG. 9B, mice were injected with human red blood cells (solid circles),cultured enucleated erythroid cells (dashed diamonds), culturedenucleated erythroid cells that express an intracellular exogenousprotein (dotted squares) and cultured enucleated erythroid cells thatexpress a surface exogenous protein (open triangles). The clearance rateof the human cells is measured as the percentage of CFSE+ cellsremaining over time, scaled to the initial time point (20 minutes postinjection). There is no significant difference in clearance rate betweenthe four samples.

These data clearly demonstrates that cultured enucleated erythroid cellshave substantially similar circulation to normal human red blood cells.Furthermore, exogenous proteins expressed either in the intracellularspace or on the surface of the cells do not substantially affect thecirculation behavior of these cells. This is an important result fortherapeutic translation of the technology.

Example 32 Assessment of Adverse Circulatory Events

The incidence of adverse events caused by cultured eyrthroid cells incirculation were assessed by detection of fibrinogen breakdown productsin blood and histology in animals injected with cultured erythroidcells.

Detection of Fibrinogen Breakdown Products. Mice were injected withcultured erythroid cells as described herein. Blood was collected frommice by submandibular puncture in an EDTA-containing tube. Cells wereseparated by centrifugation and plasma was collected. The levels offibrinogen breakdown products fibrinopeptide A and fibrinopeptide B weremeasured in mouse plasma by ELISA (MyBiosource) following manufacturer'sinstructions.

Histology. Tissue samples from the same mice were collected followingnecropsy. Tissues were trimmed, embedded in paraffin wax, and sectioned.Tissue sections were stained by H&E staining and trichrome staining.Microscope images were taken at 10× and 20× magnification.

For therapeutic applications, it is important that cultured erythroidcells and cultured erythroid cells that contain exogenous proteins(either intracellularly or on the surface) not induce adverse events,such as activation of the clotting cascade and tissue thrombusformation.

FIGS. 10A and 10B show the levels of fibrinopeptide A and B in mouseplasma for mice injected with (1) human red blood cells, (2) culturedenucleated erythroid cells, (3) cultured enucleated erythroid cellsexpressing an intracellular exogenous protein, (4) cultured enucleatederythroid cells expressing a surface exogenous protein, and (5)recombinant protein alone. The levels of fibrinopeptide A and B, amarker of fibrinogen breakdown and activation of the clotting cascade,are substantially similar for all samples.

FIGS. 10C and 10D show histologically stained sections of spleen for amouse injected with cultured enucleated erythroid cells (10C) andrecombinant protein (10D). There is no substantial difference betweenthe tissue, and no identifiable tissue damage in spleen, liver, lung,brain, heart, and kidney was observed between any of the samples.

These data conclusively demonstrate that cultured erythroid cells, withor without exogenous protein, do not induce any adverse events while incirculation in mice.

Example 33 Assessment of Exogenous Protein Retention in Circulation

The retention of exogenous proteins in and on cultured enucleatederythroid cells was assessed by flow cytometry and Western blotting.

1. Retention of Exogenous Protein Assessed by Flow Cytometry

Erythroid cells were cultured as described herein. A transgene constructencoding glycophorin A signal sequence, antibody scFv specific tohepatitis B surface antigen, HA epitope tag, and glycophorin A codingsequence was assembled by Gibson assembly as described herein. Thetransgene was introduced into the erythroid cells by lentiviraltransduction as described herein. The cells were cultured to terminaldifferentiation as described herein. Cells were fluorescently labeledwith CFSE and injected into an immunocompromised NSG mouse (JacksonLabs) via the tail vein (1×10{circumflex over ( )}8 cells per mouse). Atregular intervals blood was collected by submandibular puncture.Collected cells were stained with a fluorescent anti-HA antibody(Abcam), and analyzed by flow cytometry. Human cells were identified asCFSE+ cells, and exogenous protein retention was assessed by thefraction of CFSE+ cells that also stained positive for the epitope tag.

2. Retention of Exogenous Protein Assessed by Western Blot

Erythroid cells were cultured as described herein. A transgene constructencoding adenosine deaminase and an HA epitope tag was assembled byGibson assembly as described herein. The transgene was introduced intothe erythroid cells by lentiviral transduction as described herein. Thecells were cultured to terminal differentiation as described herein.Cells were fluorescently labeled with CFSE and injected into animmunocompromised NSG mouse (Jackson Labs) via the tail vein(1×10{circumflex over ( )}8 cells per mouse). At regular intervals bloodwas collected by submandibular puncture. Collected cells were washed,lysed, and analyzed by Western blot as described herein with a detectionantibody against the HA epitope tag.

For therapeutic applications, it is important that cultured erythroidcells that contain exogenous proteins either intracellularly or on thesurface retain these transgenes when in circulation. This feat isnon-trivial given that it is widely hypothesized in the field thaterythroid cells undergo a rigorous program of maturation and eliminationof proteins unnecessary for basic function when in circulation as theymature (Liu J et al. (2010) Blood 115(10):2021-2027, Lodish H F et al.(1975) Developmental Biology 47(1):59).

FIG. 11 shows that exogenous proteins expressed in and on culturedenucleated erythroid cells were retained in circulation. In FIG. 11A,mice were injected with cultured enucleated erythroid cells thatexpressed antibody scFv on their surface. The percentage of antibodyscFv-positive cells began and remained steadily at approximately 50%through the duration of the multi-day circulation study. In FIG. 11B,mice were injected either with cultured enucleated erythroid cells thatexpressed a cytoplasmic enzyme with an HA tag or with recombinant enzymewith an HA tag. When analyzed by Western blot, it is clear that theenzyme retained within the cultured cell for the duration of theexperiment. The decrease in band intensity is attributable to theclearance of cells during the experiment, not from the removal ofexogenous enzyme from said cells.

The data clearly demonstrate that exogenous proteins expressed in and onculture enucleated erythroid cells are retained in and on the cells incirculation, which has tremendous and unprecedented implications fortherapeutic relevance.

Example 34 Assessment of Half-Life Extension In Vivo

Erythroid cells were cultured as described herein. A transgene constructencoding adenosine deaminase and an HA epitope tag was assembled byGibson assembly as described herein. The transgene was introduced intothe erythroid cells by lentiviral transduction as described herein. Thecells were cultured to terminal differentiation as described herein.Cells were fluorescently labeled with CFSE and injected into animmunocompromised NSG mouse (Jackson Labs) via the tail vein(1×10{circumflex over ( )}8 cells per mouse). At regular intervals bloodwas collected by submandibular puncture. Collected cells were washed,lysed, and analyzed by Western blot as described herein with a detectionantibody against the HA epitope tag.

A transgene encoding adenosine deaminase with an HA tag at theC-terminus was constructed by Gibson assembly as described herein. Thetransgene was introduced into HEK-293T cells by lipofectaminetransfection (Life Technologies) as described herein. The protein waspurified from the cell culture supernatant after 7 days using an HAaffinity resin (Pierce) according to manufacturer's instructions.Protein concentration was assessed by absorbance of light at 280 nm.Protein (40 ug) was injected into an immunocompromised NSG mouse(Jackson Labs) via the tail vein. At regular intervals blood wascollected by submandibular puncture. Plasma was analyzed by Western blotas described herein with a detection antibody against the HA epitopetag.

In FIG. 11B, mice were injected either with cultured enucleatederythroid cells that expressed a cytoplasmic enzyme with an HA tag orwith recombinant enzyme with an HA tag. When analyzed by Western blot,it is clear that the enzyme's circulating half-life is significantlyextended when expressed within a circulating cell compared to wheninjected in soluble form.

Example 35 Assessment of Clearance Rate In Vivo—Platelets

A population of exogenous thymidine phosphorylase expressing plateletsis cultured using the herein detailed procedure and is labeled with CFSEand injected into an NSG mouse via the tail vein. A population of nativehuman-sourced platelets is similarly labeled with CFSE and injected intoanother mouse. Samples are taken from both mice at 10 min, 1 h, 4 h, 8h, 24 h, and 48 h and flow cytometry is used to quantify plateletcirculation levels. The half-life of natural vs cultured platelets iscompared.

Example 36 Assessment of Adverse Circulatory Events—Platelets

For therapeutic applications, it is important that cultured plateletsand cultured platelets that contain exogenous proteins (eitherintracellularly or on the surface) not induce adverse events, such asactivation of the clotting cascade and tissue thrombus formation. Uponinjection of cultured platelets into an NSG mouse via the tail vein,fibrinogen breakdown products fibrinopeptide A and fibrinopeptide B aredetected in mouse plasma by ELISA following manufacturer's protocol(MyBiosource). Tissue samples from NSG mice are collected followingnecropsy. Tissues are trimmed, embedded in paraffin wax, and sectioned.Tissue sections are stained by H&E staining and trichrome staining.Microscope images are taken at 10× and 20× magnification and assessed bya trained pathologist for any pathogenic features.

Example 37 Assessment of Exogenous Protein Retention inCirculation—Platelets

The retention of exogenous proteins in and on cultured platelets isassessed by flow cytometry and Western blotting.

CFSE labeled platelets that contain intracellular exogenous protein areinjected into a mouse via the tail vein. At regular intervals blood iscollected by submandibular puncture. Blood is centrifuged to isolate theplatelet-rich plasma, which is then lysed, and analyzed by Western blotwith staining for an epitope tag present on the exogenous protein.

Example 38 Acquisition of Donor Cells for Production

After obtaining informed consent, healthy CD34+ stem cell donors receiverhG-CSF (Granocyte or Neupogen), 10 ug/kg/day s.c., for 5 days forperipheral blood stem cell mobilization and then undergo apheresis for 2consecutive days to collect mobilized CD34+ HSC. Mononuclear cells (MNC)are isolated from mobilized peripheral blood by Ficoll density gradientcentrifugation and are split in two parts. One part is used to purifyCD34+ cells by using anti-CD34-coated magnetic beads (Miltenyi Biotec,Inc., Germany), relative to Miltenyi protocol. The purity of the CD34+fractions is controlled. CD34+− enriched HSC are then used immediatelyin the two-step culture method or frozen until use in the one-stepculture method.

Complete medium (CM) used is RPMI 1640 (Eurobio, France), supplementedwith 2 mM L-glutamine and 100 IU/ml penicillin-streptomycin (Gibco,Grand Island, N.Y., USA) and 10% heat-inactivated FBS (Gibco). IMDM(Gibco), supplemented with 10% heat-inactivated FBS, is used forexpansion. Recombinant human stem cell factor (rhSCF), thrombopoietin(TPO), fetal liver tyrosine kinase 3 ligand (Flt-3L), GM-CSF, andTNF-alpha are purchased from R&D Systems (Minneapolis, Minn., USA).

Example 39 Scale-Up for Production

Erythroid cells are scaled up in volume progressively, maintaining thecells at a density of between 1×10{circumflex over ( )}5 and2×10{circumflex over ( )}6 cells/mL in static culture. Expansion stageis seeded at 10{circumflex over ( )}5/ml and includes 3-7 progressivevolume transfers; 100 ml, 500 ml, 1 L, 10 L, 50 L, 100 L, 100 L. Duringthe course of production the cell media includes a combination of IMDM,FBS, BSA, holotransferrin, insulin, glutamine, dexamethasone, betaestradiol, IL-3, SCF, and erythropoietin. When the cells reach a volumeappropriate for seeding the production bioreactor, they are transferredto the production bioreactor for final scale-up and differentiation.

Example 40 Culturing Cells in a Bioreactor (Wave)

The WAVE Bioreactor 2/10 system is set up according to the operatormanual. In brief, the Cellbag is assembled on the rocking unit, which isplaced on the perfusion module. After inflating the bag with air, theweight is set to zero. Subsequently, the bag is filled with theappropriate amount of culturing media and incubated for at least twohours, allowing the media to reach 37 C. The media and cells aretransferred to the bag via a transfer flask, a special designed DURANglass bottle with two ports. In the upper part of the flask, a filter isconnected to the port. In the other port, by the bottom of the flask, atube is assembled. The tube one the transfer flask is coupled with thefeed connection on the Cellbag. The transfer flask is maintained in aLAF hood, to decrease the risk of contamination.

Before perfusion is started, tubing and containers for harvest and feedare connected to the Cellbag. Tubing is prepared as follows; a 50 or 70cm long Saniflex ASTP-ELP silicone tubing (Gore/Saniflex AB), with aninner and outer diameter of 3.2 respectively 6.4 mm, is equipped withmale luer lock connections in both ends. The silicone tubing isconnected to one end of a C-Flex tube, via a female luer lock. At theother end of the C-Flex tube a male luer lock is assembled and tubingsare thereafter autoclaved. Luer locks are held in place with zip-ties onall tubes. Prior to perfusion, the silicone part is connected to theCellbag and the C-Flex part to a 5 L container (Hyclone Labtainer) forboth feed and harvest. All connections are performed in a laminarairflow cabinet.

Control of environmental and metabolic factors can alter the expressionor activity of transcription factors and gene regulatory proteins oferythroid cells in culture, see e.g., Csaszar et al., 2009 BiotechnolBioeng 103(2):402; Csaszar et al. 2012 Cell Stem Cell 10(2):218. Toprovide control over inputs and outputs in the reactor a micro-volumedelivery system is created, a key component of which is a 60-80 cm longfused silica capillary (#TSP100375, Polymicro Technologies) with aninternal diameter of 100 um. At the input end, the capillary is fed witha luer-lok tip stock syringe (#309585 BD) connected via a PEEK luer to aMicroTight adapter (#P-662, Upchurch Scientific). The stock syringe isloaded on a Model 33 Twin Syringe Pump (#553333, Harvard Apparatus),kept in a refrigerator at 4 C. At the output end, the capillary entersthe bioreactor: a two port FEP cell culture bag (#2PF-0002, VueLife)placed on an orbital shaker in a cell culture incubator at 37 C with 5%CO2. The capillary is fed through a self-sealing rubber septa (#B-IIS,InterLink) with a needle, into the midpoint of the bioreactor. Theopposing connector on the bioreactor is replaced with an additionalself-sealing rubber septa. Stock syringes and delivery capillaries areblocked overnight before use with a solution of PBS with 10% fetalbovine serum to prevent protein adhesion to syringe and capillary walls.

National Instruments LabVIEW 7.1 is used to create a program to controlthe syringe pump's injections. The program's basic dosing strategy is aninitial injection to concentration L1 followed by wait time t1 andsubsequent injections, each to concentration L2 and followed by waittime t2, repeated for n times. The user inputs the flow rate, the stockconcentration, the initial culture volume, the desired concentrationafter injections, the time between injections, and the total number ofinjections.

Example 41 Assessment of Immunogenicity and Tolerance Induction

1. Tolerance Induction in Mice

In mice, tolerance can be induced by 3 sequential intravenous injectionswith a cell composition of the invention comprising an antigenicprotein, in this example ovalbumin (OVA). Naive mice are injected ondays −7, −3 and −1 with either free OVA or OVA expressed within the cellcomposition of the invention (cell-OVA). Mice are then immunized to OVAby two injections of the antigen mixed with poly I:C adjuvant(Invivogen, San Diego, Calif.) to induce a strong immune response.

2. Assessment of Antibody Titer

IgG levels in mouse serum are evaluated by standard ELISA. Briefly,serum is obtained at various time points from blood samples of mice thathave been injected with a cell composition of the invention comprisingan antigenic protein, e.g. Ovalbumin (OVA), and from mice that have beeninjected with free or recombinant OVA. A standard ELISA assay is used,with OVA as the antigen (1 μg/ml in 50 mM carbonate buffer, pH 9.7)adsorbed onto assay plates. Serum samples are serially diluted in therange of 1:50-1:200 for pre-treatment or no-treatment serum, and1:400-1:500,000 for post-treatment serum and tested in duplicate. Thebinding of anti-OVA antibodies in serum to the adsorbed recombinant OVAis detected colorimetrically with a secondary anti-mouse immunoglobulinconjugated to horseradish peroxidase, followed by treatment with achromogenic substrate.

3. Analysis of T cell Responses

Tolerance is induced to the antigenic protein OVA as described herein.Mice are euthanized 7 days after the 2nd administration of theimmunization phase injection of OVA, and their spleens are collected.Spleen cell suspensions are obtained by straining the organs through a70 micron cell strainer and after RBC lysis with a 0.8% ammoniumchloride solution (Stem-Cell Technologies, Grenoble, France). Allsamples are incubated with anti-Fc receptor antibody (purifiedanti-CD16/32, Ozyme, San Diego, CA) to prevent non-specific bindingprior to incubations with antibodies for analysis. The followingmonoclonal antibodies (Abs) are used for spleen cell staining:PC5-anti-CD62L (MEL14) and PC7-anti-CD8, purchased from Biolegend. OVApeptide-MHC tetramers (PE-Kb-SIINFEKL tetramers) are purchased fromBeckmann Coulter. OVA-specific T cells are identified by flow cytometryas cells that are double positive by staining with anti-CD8 and OVApeptide-MCH tetramers. Of this cell population, the percentage ofOVA-specific CD8 T cells that are activated is determined by thefraction of cells that are positive by staining anti-CD62L antibody.

4. In Vivo T Cell Lysis Assay

Naive spleen cells are pulsed with 10 micrograms/ml of SIINFEKL peptide(Genscript, Piscataway, N.J.) at 37 C for 1 hour and then labeled with0.4 microM CFSE (CFSE low). A control population of untreatedsplenocytes is labeled with 4 microM CFSE (CFSE high). CFSE low and CFSEhigh cells are combined in a ratio of 1:1 and 1E7 cells per mouse areinjected by i.v. route to mice that have previously been tolerized toOVA antigen as described herein or to mice that have been immunized withOVA antigen as described herein. Sixteen hours later, spleen single-cellsuspensions are prepared as described herein and analyzed using flowcytometry to determine the CFSE low/CFSE high cell ratio.

Example 42 Assess Expansion and Differentiation of Cultured ErythroidCells

It is important to assess the expansion, differentiation, andenucleation in vitro differentiated cells to ensure that theintroduction of a transgene does not negatively affect the quality ofthe cells in culture. Expansion is assessed by cell counting.Differentiation is assessed by flow cytometry, Western blot, and RT-PCR.Enucleation is assessed by flow cytometry.

Assessing Expansion Rate by Cell Counting. Erythroid cells are culturedas described herein. At various time points, cells are collected, washedwith PBS, and counted using a Countess Automatic Cell Counter instrument(Life Technologies). The expansion rate of the cells is determined bythe growth in number of cells over time.

Assessing Differentiation by Flow Cytometry. Erythroid cells arecultured as described herein. At various time points, cells arecollected, washed with PBS, and stained with 1:100 dilutions offluorescent antibodies against the cell surface markers GPA (CD235a),CKIT (CD117), and TR (CD71), purchased from Life Technologies. Labeledcells were analyzed by flow cytometry as described herein.

Assessing Differentiation by Western Blot. Erythroid cells are culturedas described herein. At various time points, cells are collected, washedwith PBS, lysed with RIPA buffer, and analyzed by Western Blot asdescribed herein using antibodies for differentiation markers GATA1,GATA2, Band3, CD44, and actin (Abcam).

Assessing Enucleation by Flow Cytometry. Erythroid cells are cultured asdescribed herein. At various time points, cells are collected, washedwith PBS, and stained with a fluorescent antibody against glycophorin A(Life Technologies) and the nucleic acid stain DRAQ5 (Pierce) atmanufacturer-recommended dilutions, and analyzed on an Attune flowcytometer as described herein.

Assessing Enucleation by Microscopy (Benzidine-Giemsa). Erythroid cellswere cultured as described herein. At various time points, cells werecollected, washed with PBS, and spun onto slides using a Cytospin(Thermo Scientific). Cells were fixed cells after cytospin with −20 Cmethanol for 2 min at room temp, rinsed with water, and air-dried. Abenzidine tablet (Sigma#D5905) was dissolved with 10 mL PBS, to which 10μL of H2O2 was added. The solution was filtered with a 0.22 um syringefilter. The cell spot on the slide was covered with 300-500 uL ofbenzidine solution, incubated at room temperature for 1 hr, then washedwith water. Giemsa stain was diluted (Sigma#GS500) 1:20 with water. Thecell spot on the slide was covered with 300-500 uL Giemsa solution,incubated at room temperature for 40 minutes, washed with water, andair-dried. Slides were then mounted and sealed before imaging on amicroscope.

FIG. 12A shows the expansion rate of erythroid cells in culture during aseven day window of expansion and differentiation for cells that containtransgenes (dashed line and dotted line) and cells that do not contain atransgene (solid line). Of note, the expansion rate of cultured cellsthat contain a transgene is indistinguishable from that of cells that donot contain a transgene.

FIG. 12B is a collection of flow cytometry plots for cells stained withantibodies against the cell surface differentiation markers GPA andCKIT. At this particular stage of differentiation, the culture is losingits CKIT expression and increasing its GPA expression as the cellsapproach terminal maturation. Of note, cultured cells that contain atransgene are indistinguishable from those that do not contain atransgene by this metric of differentiation.

FIG. 12C is a collection of flow cytometry plots for cells stained withan antibody against the surface marker GPA and a fluorescent DNA stain.Three cell populations are evident: (1) cells that are GPA-high andDNA-low, comprising enucleated erythroid cells; (2) cells that areGPA-high and DNA-high, comprising erythroid cells that still containgenetic material; and (3) cells that are GPA-low and DNA-high,comprising pyrenocytes or the membrane-encapsulated ejected nuclei fromenucleated cells. Of note, cultured cells that contain a transgene areindistinguishable from those that do not contain a transgene by thismetric of enucleation.

The introduction of a transgene into cell culture does not noticeablyaffect the rate of expansion, the differentiation, or the rate ofenucleation of the cells in culture.

Example 43 Assess Hemoglobin Content

1. Total Hemoglobin

Erythrocyte hemoglobin content was determined by Drabkin's reagent(Sigma-Aldrich, product D5941) per manufacturer's instructions. Briefly,blood cells were combined with the reagent in an aqueous buffer, mixedthoroughly, and absorbance of light at a wavelength of 540 nm wasmeasured using a standard spectrophotometer. A soluble hemoglobinstandard curve was used to quantify the hemoglobin content in the cells.

2. Hemoglobin Typing by RT-PCR

Cells were lysed and total RNA is collected. Reverse Transcription wascarried out with the SuperScript First-Strand Synthesis System forRT-PCR (Life Technologies) according to manufacturer's protocol.Briefly, total RNA (5 ug) was incubated with 150 ng random hexamerprimer and 10 nmol dNTP mix in 10 uL H2O for five minutes at 65 C then 1minute on ice. The reaction master mixture was prepared with 2 uL 10× RTbuffer, 4 uL of 25 mM MgCl2, 2 uL of 0.1 M DTT, and 1 uL of RNAseOUT.The reaction mixture was added to the RNA/primer mixture, mixed briefly,and then placed at room temperature for 2 min. 1 uL (50 units) ofSuperScript II RT was added to each tube, mixed, and incubated at 25□Cfor 10 min. The reaction was incubated at 42 C for 50 min, heatinactivated at 70 C for 15 min, then stored on ice. 1 uL RNase H wasadded and incubated at 37 C for 20 min. This reaction product, the1^(st) strand cDNA, was then stored at −20 C until needed for RT-PCRreaction.

Primers to amplify the different hemoglobin genes and control genes werepurchased from IDT-DNA. The primers were as follows:hHBB_F—tcctgaggagaagtctgccgt (Seq. ID No. 9);hHBB_R—ggagtggacagatccccaaag (Seq. ID No. 10);hHBA_F1—tctcctgccgacaagaccaa (Seq. ID No. 11);hHBA_R1—gcagtggcttagcttgaagttg (Seq. ID No. 12);hHBA_F2—caacttcaagctaagccactgc (Seq. ID No. 13);hHBA_R2—cggtgctcacagaagccag (Seq. ID No. 14);hHBD_F—gactgctgtcaatgccctgt (Seq. ID No. 15);hHBD_R—aaaggcacctagcaccttctt (Seq. ID No. 16);hHBG2_F—cactggagctacagacaagaaggtg (Seq. ID No. 17);hHBG2_R—tctcccaccatagaagataccagg (Seq. ID No. 18);hHBE_F—aagagcctcaggatccagcac (Seq. ID No. 19);hHBE_R—tcagcagtgatggatggacac (Seq. ID No. 20);h18S-RNA-F—cgcagctaggaataatggaatagg (Seq. ID No. 21);h18S-RNA-R—catggcctcagttccgaaa (Seq. ID No. 22).

An RT PCR reaction mix was prepared with 25 uL SYBR Green Mix (2×)(Applied Biosystems), 0.5 uL 1^(st) strand cDNA, 2 uL forward/reverseprimer pair mix (each primer at 5 pmol/uL), in a total volume of 50 uLH2O. Reactions were run in an ABI Prism SDS 7000 instrument (appliedbiosystems) using the following amplification cycle: 50 C 2 min, 1cycle; 95 C 10 min, 1 cycle; 95 C 15 s→60 C 30 s→72 C 30 s, 40 cycles;72 C 10 min, 1 cycle. Dissociation curve analysis and RT-PCR results wasperformed with the SDS 7000 instrument.

Example 44 Assessment Differentiation of Cultured Platelets—FACS

The differentiation state of platelets in culture can be assessed byflow cytometry. Megakaryocytes (MKs) represent a distinct cellularmorphology that precedes terminal platelet differentiation. To determinethe extent of maturation toward MKs, 1×10{circumflex over ( )}6 culturedcells (LAMA-84 and CD34+ cells) are washed and then labeled with (a)anti-CD41-FITC (GpIIb/IIIa; BD Bioscience, San Jose, Calif., USA) oranti CD71-FITC or (b) anti-CD33-FITC, anti-CD41-PE, anti-CD45-PerCp andCD34-APC (Beckman Coulter, Fullerton, Calif., USA), and analyzed for thepercentage of CD41 cells generated.

To determine the amount of ploidy, differentiated LAMA-84 cells arefixed overnight in 75% ethanol at 4° C. and labeled with propidiumiodide (PI, 50 μg/ml) and analyzed using the FACScalibur (BectonDickinson), whereas day 14 differentiated CD34+ cells are analyzedquantitatively under a microscope after May-Grunwald/Giemsa staining byquantitating the number of nuclei per cell and specific morphology ofMKs with this stain. Only cells with MK morphology are analyzed. Thepresence of multinucleated cells in the cytospin preparation isindicative of the presence of polyploid MKs. Differentiated CD34+ cellsare assessed for the presence of multinucleated mature MKs bymorphology.

Example 45 Assessment Differentiation of Cultured Platelets—qPCR

The differentiation state of platelets in culture can be assessed byquantitative PCR. Platelet RNA is extracted to further characterize thecultured cells. Total RNA is extracted using TRIzol reagent(Invitrogen). The purity of each platelet preparation is assessed by PCRanalysis of platelet (GPIIIa) and leukocyte (CD45) markers. Theintegrity of platelet RNA is assessed using Bioanalyzer 2100 (Agilent)prior to further analyses.

Total RNA is collected from cell lysate and a cDNA library is generatedusing a commercial synthesis kit (Clontech). The labeled cDNAs arequantified with the Quant-iT PicoGreen dsDNA Kit (Invitrogen) anddiluted to 3 pM for loading into a single lane and sequencing on anIllumina 1G Genome Analyzer (Solexa).

Raw sequences are filtered through serial quality control criteria.First, the presence of at least 6 nt of the 3′ Solexa adapter isverified. The sequence reads that did not comply with this criterion arediscarded, whereas the others are trimmed to remove the adapter sequenceharbored at the 3′ end. The remaining tags are further filteredregarding their length (>10 nt), copy number (>4 reads) and readability(<9 non-identified nucleotides, annotated N). Reads complying with allthose criteria are subsequently defined as usable reads.

All the usable reads are aligned to pre-microRNAs extracted from miRBasedatabase. Sequence tags that matched perfectly to more than oneprecursor are distributed equally among them. In order to account forDrosha and Dicer imperfect cleavage, any sequence tag that perfectlymatched the pre-microRNA in the mature microRNA region, allowing up to 4nt shift as compared to the reference mature microRNA position, isconsidered as a mature microRNA. The microRNA expression level isdefined as the number of reads mapping each mature microRNA normalizedto the total number of usable reads, considering that the overall numberof small RNAs is invariant. The relative abundance of each microRNA isdefined as the number of reads mapping each microRNA compared to thetotal number of reads mapping mature microRNAs.

Example 46 Purification by Centrifugation

Cultured cell fractions can be purified and separated from nuclei andcontaminating alternate-density cell types via centrifugation. Cells arecentrifuged at 200 g for 15 minutes to isolate an erythrocyte andreticulocyte rich fraction. The supernatant is pipetted off and thedesirable cell fraction is then washed in modified Tyrode's buffer(containing 138 mM NaCl, 5.5 mM dextrose, 12 mM NaHCO3, 0.8 mM CaCl2,0.4 mM MgCl2, 2.9 mM KCl2, 0.36 mM Na2HPO4 and 20 mM Hepes, pH 7.4) inpresence of 1 μM prostaglandin I2, and resuspended in the same buffer.

Example 47 Purification by Chemical Enucleation

Enucleation of cultured cells can be stimulated by chemical additives tothe culture, which can help increase the enucleated fraction of cellsprior to purification. Erythroid cells are cultured as described herein.48 hours prior to collection, cells are incubated with 210 mM Me2SO.Cells are then collected by centrifugation at 350×g for 5 min at roomtemperature, resuspended at a level of 3×105 cells per ml in freshmedium containing 210 mM Me2SO and 5 ug/mL of cytochalasin B (or otheractin or nucleus manipulating molecule, ie. p38 MAPK, psoralens) andincubated at 37 C. The proportion of cells without nuclei is assessed byflow cytometry as described herein, using DRAQS as a nucleic acid stainand antibodies against glycophorin A as an erythroid surface marker ofdifferentiation.

Example 48 Purification by Acoustophoresis

Several mechanical separation systems may be used to obtain a uniformcell population. Free flow acoustophoresis represents one mechanicalseparation method (Petersson 2007, American Chemical Society). Whilesuspended in saline solution (0.9 mg/mL) with nutrient additives,including CsCl (0.22 g/mL), is added to the saline solution. A samplesuspension containing cultured erythroid cells is processed using anacoustopheresis chip (Cell-Care) with two active outlets (flow rate 0.10mL/min per outlet).

Syringe pumps (WPI SP260P, World Precision Instruments Inc., Sarasota,Fla.) are used to control the flow rates in the chip. All outlets areindividually connected to high-precision glass syringes (1005 TLL and1010 TLL, Hamilton Bonaduz AG, Bonaduz, Switzerland) via the injectorsusing Teflon tubing, allowing independent control of the outlet flowrates. The clean fluid inlet is connected to a syringe pump and the cellsuspension inlet to a 50-mm-long piece of Teflon tubing (0.3-mm i.d.)with its other end submerged in a beaker from which the samplesuspension is aspirated at a rate defined by the difference between thenet outlet flow and the clean fluid inlet flow.

The ultrasound used to induce the standing wave between the walls of theseparation channel is generated using a 20×20 mm piezoelectric ceramic(Pz26, Ferroperm Piezoceramics AS, Kvistgard, Denmark) attached to theback side of the chip. Ultrasonic gel (Aquasonic Clear, ParkerLaboratories Inc., Fairfield, N.J.) ensures a good acoustic couplingbetween the two. The piezoelectric ceramic is actuated via a poweramplifier (model 75A250, Amplifier Research, Souderton, Pa.) connectedto a function generator (HP 3325A, Hewlett-Packard Inc., Palo Alto,Calif.). Even though the acoustic waves enter the chip from the backside, a standing wave is induced between the side walls of theseparation channel as a result of the coupling of the mechanicalvibrations along the three axes of the crystal structure.

The separation process is monitored using a standard microscope and awattmeter (43 Thruline Wattmeter, Bird Electronic Corp., Cleveland,Ohio). The process can subsequently be controlled by tuning the signalfrequency, the actuation power, and the flow rates.

The cell size distributions in the samples are analyzed using a Coultercounter (Multisizer 3, Beckman Coulter Inc., Fullerton, Calif.). Eachsample is mixed with an electrolyte (Isoton II, Beckman Coulter Inc.)and analyzed using a 100-um aperture. The level of hemolysis, i.e., theconcentration of free hemoglobin from damaged red cells, is measuredusing a photometer (Plasma/low HB Photometer, HemoCue AB, Angelholm,Sweden).

Example 49 Purification by Ex Vivo Maturation

Erythroid cells that are not fully mature can be driven to maturity byex vivo incubation in a system that mimics the natural in vivomaturation triggers.

1. Co-Culture with Stromal Cells

In the final stage of culture, erythroid cells are cultured on anadherent stromal layer in fresh medium without cytokines. The culturesare maintained at 37 C in 5% CO2 in air. The adherent cell layerconsists of either the MS-5 stromal cell line or mesenchymal stromalcells (MSCs) established from whole normal adult bone marrow (seeProckop, DJ (1997) Science 276:71) in RPMI (Invitrogen) supplementedwith 10% fetal calf serum. Adherent MSCs are expanded and purifiedthrough at least two successive passages prior to use in co-culture.

2. Culture in Fibronectin-Coated Plates

In the final stage of culture, erythroid cells are cultured in platesadsorbed with human fibronectin. To produce these plates, fibronectin(Sigma Aldrich) is reconstituted with 1 mL sterile H2O/mg of protein andallowed to dissolve for at least 30 minutes at 37° C. A small amount ofundissolved material may remain. This will not affect productperformance The fibronectin solution is diluted 100× in sterile balancedsalt solution and added to the culture surface with a minimal volume.The culture surface is allowed to air dry for at least 45 minutes atroom temperature. Excess fibronectin is removed by aspiration.

Example 50 Purification by Magnetophoresis

Strategies for separating, enriching, and/or purifying erythroid cellsby magnetophoresis are known in the art, see e.g., Zborowski et al.,2003, Biophys J 84(4) 2638 and Jin & Chalmers 2012, PLOS One 20127(8):e39491. A commercial magnetic separation system (QuadroMACS™Separator combining four MidiMACS™ separation units and LD columns,Miltenyi Biotec, Auburn, Calif.) is used for magnetic erythrocyteenrichment from HSC-derived erythrocyte cultures. Cells are deoxygenatedin a Glove-Bag™ inflatable glove chamber (Cole Parmer, Vernon Hills,Ill.), filled with nitrogen (Medipure™ nitrogen, concentration >99%,Praxair, Inc., Danbury, Conn.). Before deoxygenation, all materials andequipment including the separation system, degassed sterile buffer (PBS+2 mM EDTA +0.5% BSA), and sterile collection tubes are placed in theglove bag, which is then tightly sealed. Deoxygenated cultures areloaded directly into a MACS® LD column which was placed in theQuadroMACS™ separator kept under anoxic conditions inside an inflatableglove chamber filled with N2 gas. Cells which pass through the columncontained within the magnet are labeled as negative fraction and theyare expected to be “non-magnetic”, including HSCs and erythroid cellsbefore final maturation. The cells retained in the separation column arelabeled as positive fraction, which is “magnetic” and consist ofmaturing RBC-like cells nearly full of functional hemoglobin. They areeluted from LD column after its removal from the magnet. Once separationis finished, oxygenated cells are reversibly recovered by exposing thecollected cells to air.

Example 51 Purification by FACS

A population of erythroid cultured cells is sorted using aBecton-Dickinson Aria IIu cell sorter. Prior to sorting, cells arecollected, washed with PBS, and stained with a fluorescent antibodyagainst glycophorin A (Life Technologies) and the nucleic acid stainDRAQ5 (Pierce) at manufacturer-recommended dilutions. A 100 μm nozzle isused with a drop drive frequency of 28,000 drops/second. The samplethreshold rate is approximately 4000 events/second. The temperaturecontrol option is used to maintain sample and collection tubes at 4° C.the entire duration of sorting. Additionally, the sample agitationfeature is used at 200 rpm to prevent the sample from sedimentingthroughout the sort. The sample is sorted in aliquots of approximately750 μl dispensed from the syringe. Meanwhile, during these pauses thecollection tubes are kept at 4° C., protected from the light, and gentlymixed prior to resuming sort. The sorted samples are collected into a12×75 mm borosilicate glass collection tube containing 250 μl DMEMsupplemented with 10% FCS.

Example 51 Purification by Enzymatic Treatment of Cells

Allogeneic erythrocyte sourcing may benefit from A and B antigen removalto generate a universally compatible product. This may be facilitated bya set of enzymes capable of selectively cleaving the galactose groups,rendering the erythroid cells more immunogenically favorable.

Two types of recombinant proteins of endo-β-galactosidase, which areoriginally identified from Clostridium perfringens, are produced in E.coli BL-21 using standard cloning methods. ABase is prepared forreleasing A/B Ag and endo-β-galactosidase C (EndoGalC) for releasingGalα1-3Galβ1-4GlcNAc (Gal Ag), which is known to be highly immunogenicin xenotransplantation, and has a carbohydrate structure resembling theA/B Ag. ABase cleaves Galβ1-4GlcNAc linkage in blood type A[GalNAcα1-3(Fucα1-2) Galβ1-4GlcNAc] and in blood type B[Galα1-3(Fucα1-2) Galβ1-4GlcNAc].

Briefly, after cloning of ABase, an expression plasmid with a C-terminalHis tag is constructed in the pET-15b vector eabC without signalpeptide. This exogenous gene is transformed into E. coli BL-21 cells.The enzyme produced in the cells as a soluble protein fraction ispurified over a nickel-nitrilotriacetic acid column (QIAGEN GmbH,Hilden, Germany). Finally, 5 mL of purified recombinant ABase isobtained at the concentration of 3.6 mg/mL with the specific activity of1500 U/mg. One unit of the enzymatic activity is defined as the amountof the enzyme required to hydrolyze 1 μmol of the substrate per min.

The effect of ABase treatment on Ag presence, Ab binding and complementactivation is examined. Human A/B RBC are digested with ABase andsubjected to flow cytometric analysis after incubation withcross-reactive (anti-A or anti-B or anti-A and B containging; type B,type A or type O respectively) human sera. The mean fluorescenceintensity (MFI) is used to quantitate the expression level of blood typeA, B and Gal Ag. Digestion level is expressed as a percentage of bloodtype A or B Ag expressed on RBC after incubation in the absence ofABase.

Fresh blood type O sera are pooled from three healthy human volunteersand frozen at −80° C. to preserve endogenous complement activity untilused. Heat-inactivated (for 30 min at 56° C.) sera are used for analysisof Ab binding. RBC with and without enzyme (ABase) digestion areincubated with 50% blood type O sera (100 μL) diluted withphosphate-buffered saline containing 0.2% bovine serum albumin (PBS/BSA)for 30 min at 37° C. After washing, RBC are reacted with FITC-labeledanti-human IgG/IgM (DAKO, Glostrup, Denmark) (×30, 100 μL) for 30 min at4° C. and then subjected to flow cytometric analysis.

The inhibitory effect of enzyme treatment on complement activation isalso evaluated by the change of C3d deposition. After RBC are incubatedwith 50% human sera in the presence of complement activity for 15 min at37° C., RBC are reacted with FITC-labeled rabbit anti-human C3d Ab(DAKO, Glostrup, Denmark) (×100, 100 μL) for 30 min at 4° C. and thenapplied to flow cytometric analysis. The percentage of the control level(in the absence of enzyme) is calculated based on MFI to evaluate theinhibitory effect of enzyme treatment on Ab binding and C3d deposition.

Example 52 Purification of Platelets by Centrifugation

Platelets can be purified from mixed cell suspensions by centrifugation.Some 40 ml of whole blood is distributed in blood collection tubes withsodium citrate at 3.2% used as an anticoagulant. The tubes arecentrifuged at 400×g for 10 min. After this stage, three layers areclearly demarcated: plasma, red blood cells, and an intermediate zone.The plasma is at the top with the platelets, the red blood cells are atthe bottom because of their heavier density; and the fine, whitishintermediate zone consists of larger platelets and leukocytes and iscalled the buffy coat. Using a Jelco 18G needle, the upper portion ofplasma with platelets is drawn off, and the buffy coat is placed intotwo other tubes, this time with no additives: one tube to produce plasma(P tube) and the other to produce thrombin (T tube). Only 1.5 ml ofplasma is used to produce thrombin, to which 0.5 ml of calcium gluconateat 10% is added, with 15 min in a double boiler at 37° C. The two tubesare then centrifuged again, this time at 800×g, for the same length oftime (T=10 min). After this final centrifugation, the T tube contains athrombin-rich liquid while the P tube contains the plateletsedimentation and some red blood cells (erythrocyte-platelet clump). Thevolume is reduced at this stage by removing two-thirds of the totalplasma volume. The portion removed is platelet poor, while the remainingportion with the sedimented platelets (that are easily dispersible bystirring) is platelet rich.

Example 53 Thymidine Incorporation

Self-replication potential of a cell population can be assessed using athymidine incorporation assay known in the art, see e.g., Harkonen etal. 1991 Exp Cell Res 186L288 and Tanaka et al. 1992 PNAS 89:8928.

Briefly, uniformly 13C- and 15N-enriched thymidine [U-13C, 15N-TdR] isobtained from Martek Biosciences (Columbia, Md.), and 3 H-TdR (80Ci/mmol) is purchased from ICN Radiochemicals (Irvine, Calif.). Mediaand buffers are obtained from Fisher Scientific (Pittsburgh, Pa.). Allenzymes except phosphodiesterase are from Boehinger Mannheim(Indianapolis, Ind.). Phosphodiesterase II is obtained from WorthingtonBiochemical Corporation (Lakewood, N.J.). High-performance liquidchomatography (HPLC) solvents are from EM Science (Gibbstown, N.J.) andcontained <0.1 ppm evaporation residue.

Erythroid cells are cultured as described herein. Following the culture,cells are collected for use in the thymidine incorporation assay.

Cells are labeled with [U-13C, 15N]-TdR at 1.6 μg/ml for 18 h, with theaddition of unenriched thymidine to achieve a final thymidineconcentration of 1 μM. After they are washed with phosphate-bufferedsaline, the cells are cultured in supplemented DMEM for 6 h more before3 H-TdR is added at the indicated concentrations (0.1-10 μCi/ml) foranother 18-h incubation. Unlabeled thymidine is added to the samples tobring the final thymidine concentration to 0.13 μM, which is equivalentto the concentration of 3 H-TdR in the samples receiving 10 μCiradiolabel/ml. After removal of 3 H-TdR, the cells are incubated insupplemented DMEM for an additional 6-54 h before isolation of DNA.

DNA is extracted using the modified Puregene DNA isolation kit (GentraSystems, Minneapolis, Minn.). Based on the number of cells in thesample, a scale-up/scale-down procedure is used to determine the addedreagent volumes. For example, when 1×10{circumflex over ( )}7 cells areused, 21 μl containing 328 μg of proteinase K is added to 3 ml of celllysis solution. After mixing, the sample is left overnight at roomtemperature. The following day, 10 μg of RNase is added and the sampleis mixed and incubated for 2 h at 37 C. Protein precipitation solution(1 ml) is added, and the sample is incubated on ice for 5 min. Aftercentrifugation for 10 min at 2000 g, the supernatant containing DNA ismixed with 3 ml 100% 2-propanol and gently inverted 50 times or untilwhite threads of DNA became visible. The sample is then centrifuged at2000 g for 5 min. The resultant DNA pellet is dried for 5 min beforewashing in 3 ml of 70% ethanol and recentrifugation for 5 min at 2000 g.The final pellet is air-dried and then rehydrated in deionized H2O andquantitated by absorption at 260 nm. The same procedure is applied toCD34+ stem cells as a control for replicative ability.

Any DNA is denatured by boiling for 3 min, then chilled rapidly on ice.The enzymatic hydrolysis procedure is carried out with a DNAconcentration of 0.5 mg/ml. The following protocol describes volume ofreagent added per milliliter of DNA solution. DNA is hydrolyzed with 10μl of nuclease P1 (0.5 U/μl) and 5 μl of DNase I (4 U/μl) in 10 μl ofbuffer containing 200 mM MgCl2, 100 mM ZnCl2, and 1 M Tris, pH 7.2, for2 h at 45° C., followed by addition of 20 μl phosphodiesterase (4 mU/μl)and further incubation for 2 h at 37° C. Finally, 5 μl of 10 M ammoniumacetate (pH 9.0) and 10 μl of alkaline phosphatase (1 U/μl) are added,and the samples incubated for another 2 h at 37° C.

The digested DNA sample is filtered with a 0.22-μm nylon filter. Thissample is analyzed with the HPLC/CRI/IRMS system, using a 4.6×250 mmSupelcosil LC-18-S HPLC column (Supelco, Bellefonte, Pa.). The samesolvent system is used at 1 ml/min and a linear gradient of 5% to 25% Bin 15 min.

After separation by HPLC, the deoxynucleosides are analyzed usingchemical reaction interface mass spectrometry (CRIMS). In this process,the deoxynucleosides flow into a nebulization and desolvation systemdriven by a stream of helium, where they emerge as a dry particle beam.The 13CO2/12CO2 abundances from this in-line generated CO2 aredetermined with a Finnigan/MAT Delta S isotope ratio mass spectrometer(ThermoFinnigan, San Jose, Calif.) and its accompanying Isodat datasystem. 5-Fluorodeoxyuridine (Sigma) is used as an internal isotoperatio standard.

Isotope ratios (IR in equation that follows) for three nucleosides areobtained from each sample: T, dA, and dG. The enrichment of CO2 evolvedfrom each DNA-derived deoxynucleoside is computed by the equation(13)CO2 (per mil)=1000×(IR experimental−IR std)/IRstd. To maintain thehighest level of internal consistency and avoid any interexperimentaldrift, the isotope ratio for dG is subtracted across all experimentsfrom the isotope ratio for T. The data from the end of thestable-isotope labeling period (day 0) to the end of the washout (day 3)are evaluated.

Example 54 Quantification of Nuclear Material

The number of cells in a mixed population that contain DNA is assessedby flow cytometry using the DNA stain DRAQ5 (Pierce). Cells areincubated with the stain per manufacturer's instructions and analyzed ona flow cytometer, e.g., an Attune cytometer (Life Technologies). Thepercentage of cells above a predefined threshold of nuclear materialcontent is quantified.

Example 55 Tumorigenicity Assay In Vitro

To assess the replication potential of cells, a soft agar colonyformation assay can be performed. In brief, a base agar layer is made bymaking a 0.5% Agar+1× RPMI+10% FCS solution, all components warmed to 40C, and adding 1.5 mL of the solution to a 35 mm petri dish. The agar isallowed to solidify for 30 min at room temp before use.

The top agarose layer is prepared by melting 0.7% agarose in a microwaveand cooling to 40 C. A 2× RPMI+20% FCS solution is heated to 40 C. Cellsare counted and prepared for plating at 5000 cells per plate at adensity of 200,000 cells per mL. 0.1 mL of cell suspension is added to10 mL tubes, followed by 3 mL of the warm 0.7% Agarose and 3 mL of thewarm RPMI/FCS solution. The solution is mixed gently by swirling andadded (1.5 mL) to each of three or four replicate base agar plates.

Plates are incubated at 37 C in a humidified incubator for 10-30 days.Cells are fed 1-2 times per week with cell culture media, 0.75 mL/plate.

To assess colony formation, plates are stained with 0.5 mL of 0.005%Crystal Violet for >1 hr. Colonies are counted using a dissectingmicroscope.

Example 56 Tumorigenicity Assay In Vivo

Terminally-differentiated cultured erythroid cells are implanted invarious animal models to evaluate the potential for tumorigenicity.Several tissues are collected from the various models and analyzed withhistological, immunochemical, and fluorescent assays to quantifytumorigenicity.

Animals receive daily intraperitoneal injections of CsA (10 mg/kg,Sandimmune, Novatis Pharma, Nurnberg) starting two days before grafting.For the depletion of NK cells, some rats receive, in addition to CsAintraperitoneal injections of the monoclonal antibody (mAb),anti-NKR-P1A (clone 10/78, mouse IgG₁, BD Biosciences, Heidelberg,Germany) or the respective isotype control (clone PPV-06, mouse IgG₁,Exbio, Prague, Czech Republic). The anti-NKR-P1A mAb (clone 10/78) isdirected against the same epitope as the mAb (clone 3.2.3). One mg ofthe respective antibodies are given one day before the injection oferythroid cells followed by 0.5 mg at day 4 after cell transplantation.

Blood samples are taken before starting these experiments, at day 0 and4 days after erythroid cell transplantation, and at autopsy (day 92) inorder to determine the proportion of NK cells in the blood by flowcytometry. For the analysis of subcutaneous tumor growth erythroid cellsare injected in 100 μl phosphate-buffered saline (PBS) into the flank ofthe animals. Tumor growth is monitored every second day by palpation andsize is recorded using linear calipers. Animals are sacrificed beforeday 100 when a tumor volume of 1 cm³ in mice and 5 cm³ in rats isreached, when a weight loss of more than 10% occurs, or when anybehavioral signs of pain or suffering are observable. Autopsies of allanimals are performed.

Murine tissue near the site of injection is immediately frozen in liquidnitrogen or placed in phosphate-buffered 4% formalin for 16 h and thenembedded in paraffin. Spleens and lymph nodes are removed for subsequentimmunological analyses. The transplantation of erythroid cells into thestriatum of unilaterally 6-OHDA-lesioned rats is performed. Theseanimals are sacrificed 6 weeks after transplantation.

Animal tissue is analyzed by flow cytometry. Appropriate fluorescent andPE-conjugated antibodies against established cancer cell biomarkers ofCD133, CD3, CD, CD16, CD19, CD20, CD56, CD44, CD24, and CD133 are addedto the excised tissues samples and analyzed to quantify tumorigenicpotential.

Example 57 Deformability by EKTA

Erythroid cells cultured as described herein are assessed fordeformability characteristics relative to natural erythrocyte samplesvia ektacytometry.

The ektacytometer consists of a Couette-type viscometer combined with ahelium-neon laser used to produce a diffraction image of red cellssuspended in a viscous fluid between the two cylinders. When theviscometer rotates, normal red cells elongate in the shear field,causing the diffraction image to become elliptical. The ellipticity ofthe image is measured by quantifying the light intensity along the major(A) and minor (B) axes of the diffraction pattern and expressing this asa ratio (A−B)/(A+B), the deformability index (DI) or elongation index(EI). The viscosity of the medium is chosen to be greater than theinternal viscosity of the densest erythroid cells. A 31 g/liter solutionof polyvinylpyrrolidone (PVP), mw=360,000, in a phosphate buffer of 0.04M composed of K2HPO4 and KH2PO4 in distilled water yields a viscosity of0.20 poise at 25° C. and 12 poise at 37 C.

Osmolarity is adjusted with NaCl to the desired level and measured in aRoebling freezing-point osmometer. The final pH is varied by using smalladditions of 1-M solutions of NaOH and HCl and is measured in aTechnicon BG I1 blood gas analyzer. Sodium azide is added as apreservative to stock solutions to obtain 0.4 g/l.

The ektacytometer collects three primary metrics from the erythroid cellsamples and compares them to native erythrocytes; Osmolality minimum(O_(min)), deformability index (Di_(max)), and the osmolality at whichthe DI reaches half of its maximum value (O_(hyp)).

O_(min) is related to the surface area to volume ratio of the cell andhas been found to equal the 50% hemolysis point in the classical osmoticfragility test.

Di_(max) is the maximum value of the deformability index, normallyreached at 290 mosmol (the physiologic osmolality value). This indicatesthe maximum deformability of the cell under shear stress and is relatedto a number of factors, such as surface area, volume, internalviscosity, and mechanical properties of the cell membrane.

O_(hyp) is the osmolality at which the DI reaches half of its maximumvalue. This gives an indication of the position of the hypertonic partof the curve, which is related to the internal viscosity of the cell aswell as mechanical properties of the membrane, such as how it will bendunder force (stiffness).

The parameters obtained for the cultured erythroid cells are compared tothe same values for primary erythroid cells.

Example 58 Deformability by LORCA

The deformability of purified cRBC populations is measured by a laserdiffraction technique (LORCA, laser-assisted optical rotational cellanalyzer, R&R Mechanotrics). In brief, a highly diluted suspension ofcells is sheared in a Couette system with a gap of 0.3 mm between 2cylinders, one of which is able to rotate to induce shear stresses. Alaser beam is passed through the suspension, and the diffraction patternis measured at 37° C. At low shear stress, the cells are circular disks,whereas at high shear stress, the cells become elliptical. The celldeformability is expressed in terms of the elongation index (EI), whichdepends on the ellipticity of the deforming cells. Aliquots containing12.5 uL of pelleted RBC pellets are diluted in 5 mL ofpolyvinylpyrrolidone solution (molecular weight 360 000). The EI valuesat 30 Pa (referred to as Elmax) and 3 Pa are selected as representativevalues of the deformability for easy comparison between samples atvarious shear stresses.

Example 59 Assessment of Vascular Occlusion—Ex Vivo Rat Vasculature

The potential for vascular occlusion of erythroid cells can be assessedwith isolated artificially perfused rat vasculature using methods knownin the art, see e.g., Kaul et al. 1983, J Clin Invest 72:22. Briefly, inanesthetized (sodium pentabarbitol 30 mg/kg) rats of the Wistar strain,120-150 g, the right ileocolic artery and vein are cannulated withheparinized (100 uL/mL) silastic tubing at a site 3 cm distant from theileocolic junction. Under a steady-state perfusion with Ringer'scontaining 1% bovine serum albumin, the ascending colon and terminalileum (3 cm each) are sectioned between ties. After hemostatic ties ofall vascular connections is achieved, the tissue is isolated. Theisolated mesoappendix is gently spread on an optically clear Luciteblock on a microscope stage. The entire preparation is covered with aplastic saran wrap except for outlets of cannulas and the microscopeobjective.

The control arterial perfusion pressure (Ppa) and venous outflowpressures (Pv) are kept constant at 80 and 3 8 mmHg, respectively, andmonitored via Statham-Gould P-50 pressure transducers (StathanInstruments Inc, Oxnard Calif.). The venous outflow (Fv) rate ismonitored using a photoelectric dropcounter and expressed in mL/min Alapse of 10-12 min is allowed for tissue equilibration and stabilizationof Fv. Only preparations exhibiting mesoappendix microvasculature freeof host blood cells and with a steady Fv of 4.6+/−0.5 (mean+/−SD) areused. The experiments are done at 37 C.

Erythroid cells are isolated as described herein. After controlmeasurements of Ppa and Fv, erythroid cells (0.2 mL, Hct 30%) are gentlydelivered via an injection port 15 cm distal to site of arterialcannulation, and the changes in Ppa and Fv are recorded on the stripchart of a Grass polygraph (Grass Instrument Co, Quincy Mass.). Thetissue preparations are perfused for 10-15 min before the infusion ofsamples with Ringer's solution to allow stabilization of the tissue andclear the vasculature of the remaining blood cells of the host animal.The resulting obstruction after the infusion of cells can be cleared andthe flow restored by briefly (2-3 min) perfusing the vasculature withfully-oxygenated Ringer's solution at high pressure (100 mmHg).

At the end of each experiment the entire tissue preparation (free ofcannulas and luminal content) is weighed. Peripheral resistance units(PRU) are calculated and expressed as PRU=ΔP/Q=mmHg/mL/(min-g) where AP(mmHg) is the arteriovenous pressure difference and Q (mL/min-g) is therate of venous outflow per gram of tissue.

In each experiment, pressure-flow recovery time (Tpf) is determinedfollowing the bolus infusion of samples. Tpf is defined as the time(seconds) required for Ppa and Fv to return to their base-line levelsfollowing the delivery of a given sample, and it represents totaltransit time throughout the mesoappendix vasculature. The parametervalues obtained for cultured erythroid cells are compared to the valuesobtained for primary erythroid cells.

Example 60 Assessment of Vascular Occlusion—In Vitro Flow Chamber

Methods to assess vascular occlusion of erythroid cells using in vitrograduated height flow chambers are known in the art, see e.g., Zennadiet al 2004, Blood 104(12):3774.

Briefly, graduated height flow chambers are used to quantitate theadhesion of erythroid cells to endothelial cells (ECs). Slides coatedwith ECs are washed with Hanks balanced saline solution (HBSS) with 1.26mM Ca2+, 0.9 mM Mg2+ (Gibco, Grand Island, N.Y.) warmed previously to37° C. and then fit into a variable height flow chamber. The flowchamber is mounted on the stage of an inverted phase contrast microscope(Diaphot; Nikon, Melville, N.Y.) connected to a thermoplate (Tokai Hit,Fujinomiya-shi, Japan) set at 37° C. Cells are observed using a videocamera (RS Photometrics, Tucson, Ariz.) attached to the microscope andconnected to a Macintosh G4 computer (Apple, Cupertino, Calif.).Erythroid cells are cultured as described herein, and labeled withfluorescent dye PKH 26 red fluorescent cell linker kit (Sigma) followingthe manufacturer's instructions. Cells (3 mL) suspended at 0.2%(vol/vol) in HBSS with Ca2+, Mg2+ are infused into the flow chamber andallowed to adhere to the slide for 15 minutes without flow. Beforeexposure to flow, a minimum of 3 fields at each of 7 different locationsalong a line oriented normal to future flow are examined for the totalnumber of fluorescent cells. Fluid flow (HBSS with Ca2+, Mg2+) is thenstarted using a calibrated syringe pump. After exposure to flow, thefields are again examined and the number of adherent cells counted. Thefraction of adherent cells is presented as follows: Number of cellsattached after exposure to flow/Cells present per field before flow. Thewall shear stress is calculated as follows: τw=(6 μQ)/(wH [x]2), inwhich τw indicates wall shear stress (dyne/cm2); Q, volumetric flow rate(cm3/s); μ, media viscosity; w, width of the flow channel; and H(x),height of the flow chamber as a function of position along themicroscope slide.

Example 61 Assessment of Vascular Occlusion—Intravital Microscopy

Methods to assess vascular occlusion of erythroid cells using intravitalmicroscopy are known in the art, see e.g., Zennadi et al. 2007 Blood110(7):2708.

Briefly, general anesthesia of a test animal is achieved byintraperitoneal injection of 100 mg/kg ketamine (Abbott Laboratory,Chicago, Ill.) and 10 mg/kg xylazine (Bayer, Shawnee Mission, Kans.). Adouble-sided titanium frame window chamber is surgically implanted intothe dorsal skin fold under sterile conditions using a laminar flow hood.Surgery involves carefully removing the epidermal and dermal layers ofone side of a dorsal skin fold, exposing the blood vessels of thesubcutaneous tissue adjacent to the striated muscles of the opposingskin fold, and then securing the 2 sides of the chamber to the skinusing stainless steel screws and sutures. A glass window is placed inthe chamber to cover the exposed tissue and secured with a snap ring.Subsequently, animals are kept at 32° C. to 34° C. until in vivo studieswere performed 3 days after surgery.

Anesthetized animals with window chambers are placed on the stage of anAxoplan microscope (Carl Zeiss, Thornwood, N.Y.); temperature ismaintained at 37° C. using a thermostatically controlled heating pad.All infusions are through the dorsal tail vein. Erythroid cells arecultured as described herein. Cells are then labeled with Dil or DiO(Molecular Probes, Eugene, Oreg.) dyes per manufacturer's instructions.Labeled cells (300 μL; hematocrit [Hct] 0.50 [50%] in PBS with Ca2+ andMg2+) are infused, and RBC adhesion and blood flow dynamics are observedin subdermal vessels for at least 30 minutes using LD Achroplan 20×/0.40Korr and Fluar 5×/0.25 objectives. Microcirculatory events and celladhesion are simultaneously recorded using a Trinitron Color videomonitor (PVM-1353 MD; Sony, Tokyo, Japan) and JVC video cassetterecorder (BR-S3784; VCR King, Durham, N.C.) connected to a digital videocamera C2400 (Hamamatsu Photonics KK, Hamamatsu City, Japan). Thirtysegments of venules are examined for each set of conditions. Arteriolesare distinguished from venules based on (1) observation of divergentflow as opposed to convergent flow; (2) birefringent appearance ofvessel walls using transillumination, which is characteristic ofarteriolar vascular smooth muscle; and (3) relatively straight vesseltrajectory without evidence of tortuosity.

Measurement of red cell flux and adhesion is performed by examiningvideotapes produced using ×20 magnification. Cell adherence isquantitated by considering cells attached to the vessel walls andimmobile for 1 minute. The percentage of the length of vessels withdiameters up to 25 μm or more than 25 μm, occupied by SS RBCs, isquantified as follows: % venular length occupied by SS RBCs=(length ofvessel wall with adherent cells/total length of the vessel segmentsanalyzed)×100. Changes in RBC flux are calculated as follows:flux=number of circulating fluorescent human RBCs crossing a singlepoint marked on vessels less than 50 μm in diameter per minute.

Example 62 Assessment of Vascular Occlusion—Platelets

Methods to assess vascular occlusion of platelets using human vascularendothelial cells (HUVECs) can be adapted from similar methods foreythroctyes. Briefly, a 2-mL volume of 0.05% hematocrit suspension isadded to confluent HUVECs on tissue culture Petri dish. Thecone-and-plate apparatus is assembled within 1 min after addition ofplatelets and placed on a Nikon Diaphot-TMD inverted-phase contrastmicroscope (Southern Micro Instruments, Atlanta, Ga.). The motor isstarted to turn the cone, and adherence is continuously monitored at 0.1or 1 dyne/cm2 shear stress for 30 min. Temperature is maintainedconstant at 37° C. by an air curtain incubator (Nicholson PrecisionInstruments, Inc., Bethesda, Md.) blowing on the adhesion apparatus.Platelet adherence is visualized and recorded every 5 min by focusing on8 different fields of view for 20 sec per field for each time point. Theentire experiment is viewed under 400× total magnification through aCCD-72 series camera (Dage-MTI, Inc., Michigan City, Ind.) and recordedon videotape with a SVO 2000 video cassette recorder (Sony Electronics,San Jose, Calif.). Adherence is quantified off-line at the end of eachexperiment by counting individual adherent cells during manual playbackof recorded video images. The cell counts in 8 fields for each timepoint are averaged and normalized to adherent red cells per squaremillimeter of endothelium.

Example 63 Assessment of Mass/Volume/Density with Resonator

A dual suspended microchannel resonator (SMR) system is used tocharacterize the mass, volume, and density of a population ofterminally-differentiated erythroid cells based on Bryan et al, LabChip, 2014. At the start of a cell density measurement, the system isfirst flushed with filtered Percoll media, which serves as the highdensity fluid. Next, the sample bypass is filled with a dilute cellsample, and the vial heights at the sample inlet and outlet are adjustedto direct fluid flow into the first SMR. Pressure at the high densityfluid inlet is used to set the density of Fluid 2, and pressure at thewaste outlet controls the overall flow speed in the device. To minimizethe likelihood of size biasing due to heavier cells settling at thebottom of the sample vial or tubing, a fresh sample is introduced atregular intervals by flushing the sample bypass channel Data is acquiredvia LabVIEW and processed with MATLAB.

Cell concentration is monitored using a Coulter counter. Cellmeasurements are performed on cultures grown to 5×10{circumflex over( )}5-1×10{circumflex over ( )}6 cells/ml. High density fluid introducedfor measurement in the second SMR is formulated as a solution of 50%(v/v) Percoll (Sigma), 1.38% (w/v) powdered L15 media (Sigma), 0.4%(w/v) glucose, 100 IU penicillin, and 100 μg mL—1 streptomycin. Media pHis adjusted to 7.2. This Percoll media is stored at 4° C. and filteredimmediately prior to use in the dual SMR.

Example 64 Assessment of Phosphatidyl Serine Content by Annexin V

Erythroid cells are cultured as described herein. 50 μL cell suspensionis washed in Ringer solution containing 5 mM CaCl₂ and then stained withAnnexin-V-FITC (1:200 dilution; ImmunoTools, Friesoythe, Germany) inthis solution at 37° C. for 20 min under protection from light. Cellsare washed and stained by flow cytometry as described herein, andannexin-V fluorescence intensity is measured with an excitationwavelength of 488 nm and an emission wavelength of 530 nm. Relativephosphatidyl serine exposure is assessed from annexin-V fluorescence.

Example 65 Assessment of Lipid Content by Chromatography

Lipids are extracted from washed exogenous antigen-expressing EHCs bythree extractions with methanol-chloroform 1:1 at room temperature inthe presence of the antioxidant BHT (Sigma Aldrich). The pooled extractsare washed with 0.05 M KCl in the method of Folch, Lees and SloaneStanleyl957, J Biol Chem 226:497. Briefly, for the first extraction, 15mL methanol containing 0.05 mg/mL BHT are added to the washed complexesin a centrifuge tube and allowed to stand for 30 min with occasionalstirring to break up sediment. 15 mL of chloroform is then added and themixture is allowed to stand for 30 min with occasional stirring to breakup clumps. The tubes are centrifuged for 5 minutes at 1500 g and thesupernatant fractions decanted into separatory funnels fitted withTeflon stopcocks. The second and third extractions are performedsimilarly with 15 mL of the methanol-BHT added to the residue followedby 15 mL of chloroform, except the extracts stand for only 10 minuteswith occasional stirring after each addition. After centrifugation, thesupernatant fractions are pooled in a separatory funnel then 48 mL ofchloroform and 28 mL of 0.05 M KCl are added and mixed. The mixture isallowed to stand overnight in darkness at 4 C for phase separation.After being rewarmed to room temperature, the lower of the two clearphases is collected and evaporated to dryness in vacuo at 40 C in arotary vacuum evaporator. The lipid is transferred quantitatively to a10 mL volumetric flask with chloroform and stored at −22 C.

The concentration of free cholesterol in the lipid extract is determinedas follows. The lipid extract is chromatographed on a 0.5 mm layer ofSilica Gel HR (Brinkmann Instruments, Inc., Westbury, N.Y.) inhexane-diethyl ether-glacial acetic acid 80:20:1, the TLC plate isstained by spraying with 2,7-dichlorofluorescein solution (see below),the free cholesterol spot is scraped into a conical centrifuge tube andextracted once with 2.0 ml and three times with 1.0 ml of chloroform,the extract is evaporated to dryness in vacuo at 40° C. in a rotaryvacuum evaporator, and the cholesterol is estimated by the ferricchloride method of Mann 1961 Clin Chem 7:275 without saponification. Afree cholesterol standard, prepared from a commercial certified reagentgrade material by isolation through the dibromide derivative (see e.g.,Fieser J Amer Chem Soc 1953 75:5421), is taken through thechromatographic procedure and estimated with each set of determinations.The values for free cholesterol are corrected in each determination forthe recovery of the standard, which averaged 95%. The TLC is necessaryto remove the BHT, which otherwise interferes with the ferric chloridemethod by producing a brown product that absorbed at 560 nm.

The phospholipid distribution is determined in triplicate by TLC ofaliquots of the total lipid extract at 4° C. on Silica Gel HR, 0.5 mmthick, in chloroformmethanol-glacial acetic acid-water 25:15:4:2 towhich is added BHT at a concentration of 50 mg/100 ml to preventautoxidation during chromatography; the TLC plates are prepared withwater (“neutral” plates). Use of a “wedged-tip technique” for applyingthe lipid sample at the origin of the plate (see e.g., Stahl 1965Thin-Layer Chromatography, Academic Press Inc.) results in excellentseparations of the individual phospholipids. In particular, the methodprovides complete separation between phosphatidyl ethanolamine (PE),phosphatidyl serine (PS), lecithin, and sphingomyelin; a discrete spotmigrates between PS and lecithin that is identified as phosphatidylinositol (PI). The spots are made visible in UV light by spraying with asolution of 2,7-dichlorofluorescein (33.3 mg/100 ml of aqueous 2 mMNaOH) and then scraping directly into Kramer-Gittleman tubes, where thephospholipids are digested at 190° C. for 60 min with 1.0 ml of 70%perchloric acid. The remainder of the procedure is performed asdescribed above, except that after color development, the silica gel isremoved by centrifugation at 3000 g for 5 min and the absorbancy isdetermined on the clear supernatant solution. Corrections are made forthe absorbancy of corresponding areas of blank lanes.

Gas-liquid chromatography is performed on hexane-dissolved samples witha Barber-Colman instrument, model 5000, equipped with paired 8-ftcolumns of EGSS-X (an ethylene glycol succinate polyester combined witha silicone) 8% on Gas-Chrom P, 100-120 mesh (Applied ScienceLaboratories Inc.) and dual flame ionization detectors. The nitrogenflow rate is 50 ml/min at the inlet. The column temperature ismaintained at 1650 C for 10 min after injection of the sample, thenincreased at 2 C/min to 200° C.

Example 66 Assessment of Membrane Viscosity

The membrane viscosity of a population of cells can be assessed byfluorescence photobleaching assay. A 0.5-ml sample of erythroid cells iscollected and washed once in HEPES-buffered saline (132 mM NaCl, 4.7 mMKCl, 2.0 mM CaCl2, 1.2 mM MgSO4, 20 mM HEPES, adjusted to pH 7.4). Thepacked cells are then washed once in 145 mM NaCl-10 mM NaHCO3, pH 9.5,and resuspended in the same buffer with 1 mg/ml DTAF (obtained fromResearch Organics, Cleveland, Ohio). The cells are incubated on ice for1 h, then washed twice in 50 mM glycine-95 mM NaCl-10 mM NaHCO3, pH 9.5,to remove any dye that has not bound covalently to protein. Finally, thecells are washed twice and resuspended to −2% hematocrit inHEPES-buffered saline with 1 mg/ml bovine serum albumin. The sametreatment is applied to control native erythrocytes.

The flow chamber is mounted on the stage of a Leitz Diavert (Rockleigh,N.J.) inverted microscope equipped for incident-light fluorescencemicroscopy. The dichroic mirror and excitation/emission filters are thestandard combination for use with fluorescein dyes (Leitz designation12), with excitation wavelength in the range 450-490 nm. The objectiveis an oil immersion type with 100x magnification and 1.25 numericalaperture. A 100 watt high pressure mercury arc lamp (Osram, Munich) withan appropriate power supply and housing (Oriel, Stamford, Conn.) servesas the fluorescence excitation source.

A computer-controlled electronic shutter (Vincent Associates, Rochester,N.Y.) limits the exposure duration and is synchronized with aphoton-counting electronic system for measuring fluorescence intensity.The field diaphragm of the incident light illuminator is used to limitexcitation to a circular area of diameter 20-40 um. At regularintervals, an output pulse from the computer causes the shutter to openfor a typical duration of 20 ms. Light from the brief fluorescent imageis split with a series of prisms so that half the light is directed to alow-light-level SIT video camera (Model 66-SIT, Dage-MTI, Michigan City,Ind.) and half to a photomultiplier tube (Model 8850, RCA, Harrison,N.J.) enclosed in an ambient temperature housing. During the time thatthe electronic shutter is open, a video image processor (Model 794,Hughes Aircraft, Carlsbad, Calif.) is triggered to acquire thefluorescent image, providing a video snapshot that can be monitored toensure that the subject remains in focus and that no foreign objectintrudes into the field of view. Distances on the video screen aremeasured with a video caliper and calibrated by comparison with thevideo image of a stage micrometer. Also during the time the shutter isopen, the photomultiplier signal is processed with the photon-countingtechnique. An amplifier/discriminator (Model AD6, Pacific Instruments,Concord, Calif.) generates a digital logic pulse for each signal pulseabove a given magnitude, and those digital pulses are counted on a100-MHz gated counter (Model 770, EG&G Ortec, Oak Ridge, Tenn.). Themicrocomputer controls the gating, resetting, and recording of thephoton count.

A typical experiment consists of a number of preliminary fluorescencemeasurements made during brief (20 ms) pulses of excitation light,followed by an extended period of illumination (typically 30 s) duringwhich the samples cells are bleached, followed by another series ofbrief exposures, every 15-30 s, until the fluorescence appears to havecompleted its recovery.

The recovery time and other parameter values obtained for culturederythroid cells are compared to the same values obtained for primaryerythroid cells.

Example 67 Assessment of Mean Corpuscular Volume with Advia HematologyAnalyzer

The Mean corpuscular volume (MCV) of the cultured erythroid cells ismeasured using electrical impedance in an Advia 120 hematology analyzer(Siemens Healthcare). The results are compared to that of natural humanerythrocytes.

Example 68 Pathogen Testing of Cultured Erythroid Cells

RT-PCR is used to quantify adventitious virus presence in culturederythroid cell populations and confirm non-contamination (Assay No.003000.BSV, BioReliance). Sterility testing of unprocessed and finalbulk, final vials, prebanking cells, and cell and virus banks isperformed by directly inoculating the erythroid population into 2different types of media that support the growth of aerobic andanaerobic bacteria respectively. Samples are incubated for 14 daysfollowed by testing for microbial contaminants per BioReliance SterilityTesting protocol USP 71.

Example 69 Assessment of Osmotic Fragility

Osmotic fragility is evaluated to measure the resistance of theerythroid cells to lysis when exposed to hypotonic solutions. Solutionsof NaCl in water were made at concentrations spanning 0% to 1%. Cellswere incubated in each of the salt solutions for 15 minutes. The sampleswere centrifuged to pellet intact cells. Supernatant was assayed forhemoglobin content by absorption of light at 540 nm using aspectrophotometer. The point at which 50% hemolysis occurs is calculatedand compared to the value obtained for primary erythrocytes.

Example 70 Assessment of Rosetting/Immunogenicity

The direct antiglobulin test, also known as Coombs test, assesses theagglutination or resetting of erythroid cells caused by the binding ofpolyclonal antibodies from serum to surface antigens on the cell. It canbe performed with pooled human serum for general allogeneicimmunogenicity assessment, or with serum from the intended recipient forspecific immunogenicity prediction.

In brief, add 1-2 drops of cells stored in an EDTA tube to a reactiontube. Wash this tube three times with isotonic saline. After the thirdwash, prepare a 3% suspension from the washed cells. Label 2 tubes A andB. Add one drop of the washed 3% suspension to each tube. Wash thesetubes one more time. When decanting, position the tubes so that the cellbutton is on top. This will prevent too many cells from being lost inthe washing process. Drain well, and blot dry with a biowipe Immediatelyadd one drop human test serum to both tubes, and shake to mix. Allow theB tube to incubate at room temperature 5 minutes. Centrifuge the A tubefor the time calibrated for the Coombs spin on the serofuge. Immediatelyresuspend gently and examine for agglutination using the lightedagglutination viewer (Beckton Dickinson). If the A tube is positive, itis not necessary to read the B tube nor is it necessary to examine the Atube microscopically. If the A tube is negative by lighted agglutinationviewer, examine for agglutination under the microscope. If the A tubewas negative through the microscopic reading, centrifuge the B tubeafter its incubation period and repeat steps 2-4 with the B tube sample.If the B tube is negative as well, add one drop of IgG-coated CoombsControl Cells (Check Cells) to the tube and centrifuge. Examine foragglutination. Agglutination should be present in this step, or the testis invalid.

If there is no agglutination in any of the steps before addition of thecheck cells (ccc), the test is interpreted as negative. If agglutinationis observed in any of the steps before addition of the check cells, thetest is interpreted as positive.

Example 71 Assessment of Oxygen-Binding Capacity

Equilibrium oxygen binding curves at 37° C. are determined in atonometer linked to a 1-cm path length cuvette. Spectral measurementsare performed with a spectrophotometer (Cary 50; Variant Inc), and thetemperature is controlled with a Peltier module. Analyses are performedin 50 mM bis-Tris buffer (pH 7.2) containing 140 mM NaCl and 2 mMglucose. After thorough deoxygenation under nitrogen, the red cellsuspensions are equilibrated at different partial pressures of oxygen byinjection of known volumes of pure oxygen into the tonometer through arubber cap with a Hamilton syringe. The fractional saturation isestimated by simulation of the absorption spectra in the visible andSoret regions as a linear combination of the fully deoxygenated andoxygenated spectra of the RBC suspension by least squares regression.

Example 72 Assessment of Metabolic State of Cells

The erythroid cell population may be verified as metabolically activeusing a variety of different enzyme based assays to quantify importantmetabolic end products. Active glycolysis is a crucial metabolic pathwayto assess and may be measured with the following assay (Glycolysiscell-based assay kit, Cayman Chemical, Item 600450).

450 ul of assay buffer is aliquoted into a test tube, followed by 50 uLof the L-Lactic acid standard asnd mixed thoroughly. A titration curveis constructed using the lactic acid concentration standard, beginningwith a 1 mM dilution.

Cells are added to a 96 well plate and centrifuged at 1000 RPM for 5minutes. 100 uL of the standards are transferred into a separate 96 wellplate. 90 uL of assay buffer is then added to each well. 10 ul ofsupernatant in each cell well is then transferred to corresponding newwells. Add 100 ul of reaction solution to each well using a repeatingpipettor. The plates are then incubated on an orbital shaker for 30minutes at RT. The absorbance isread at 490 nm with a plate reader.Results are compared to natural cells to identify any metabolicdifferences.

Example 73 Assessment of Platelet Aggregation

Aggregation propensity of cultured or primary sourced platelets can bemonitored. Platelets are submitted to swirling analysis by shaking themin front of a light source, with the results expressed as presence orabsence of birefringence. The units of platelet concentrates producedwith a volume of 50-70 mL are left to rest for one hour and placed in alinear shaker (C-Mar®) at 70 rpm at a controlled temperature of 22±2° C.(71.6±3.6° F.).

The tests of platelets concentrates (platelet count, plateletaggregation and pH) are carried out on days 1, 3 and 5 after processing;a leukocyte count is performed only on day 1 and the microbiologicalcontrol is performed only on the 5th day of storage. In order to obtainaliquots from samples of platelet concentrates, a sterile connection(Haemonetics®) is used which ensured the integrity of the environment.Platelet aggregation is achieved using the turbidimetric aggregometrytechnique using a dual-channel Chronolog (Crono-Log Corporation®) withinfour hours of blood collection. For this, the cells are initiallyobtained through light centrifugation at 1000 rpm for five minutes, andthen centrifuged at 3000 rpm for fifteen minutes (Eppendorf®). Samplesare subjected to a platelet count in an automatic counter (HumanCount®).

After adjusting the platelet concentration, aggregation is evaluatedusing different concentrations of inducing agonists: collagen 2.0 μg/mLand ADP 7.0 μg/mL (Crono-Log Corporation®). For each test, 400 μL of PRPand 400 μL of PPP are used, each one in a different cuvette afterwaiting for spontaneous aggregation. The aggregation curve is observedafter five minutes of stimulation by inducing agonists, and soon after,aggregation is measured and expressed as a percentage according to thecurves formed during the tests. The result of the test is commonlyexpressed as a percentage of aggregation by the quantity of lighttransmitted through the test solution; aggregation is classified asnormal, low or high.

Example 74 Autologous Culture Process

The culture of erythroid cells using autologously sourced progenitorCD34+ cells is done to optimize cell immunocompatibility for patients.CD34+ cells from the bone marrow are mobilized to the periphery in apatient using GM-CSF as described herein. Between 10{circumflex over( )}6-10{circumflex over ( )}8 CD34+ cells are collected and culturedusing the aforementioned 22 day protocol using defined media. During Day4 the cells are transfected with a lentiviral vector containing a genethat codes for the expression of a therapeutic agent. Upon completion ofthe culturing protocol, the cells are purified and assessed acrossseveral quality control metrics including physical properties thatcorrelate with circulation viability, immunogenicity, replicativepotential, purity, and therapeutic dose. The cells are then stored inappropriate stabilizing solution and formulated in a syringe orappropriate delivery vehicle. The cells are then infused into the samepatient that donated the initial CD34+ cells.

Example 75 Autologous Loading Process

For the preparation of therapeutic erythroid cells loaded with asuitable exogenous antigen, autologously sourced erythrocytes can beused to optimize cell immunocompatibility for patients. Blood is drawnfrom the patient and centrifuged at 5000 g for 20 minutes. The buffycoat is removed and the remaining red cells are re-suspended inanticoagulant buffer at a density of 10{circumflex over ( )}8 cells/ml,giving a total of 10{circumflex over ( )}10 cells. The cells are loadedwith a therapeutic exogenous antigen of interest by one of the methodsdescribed above. Upon completion of the loading protocol, the cells arepurified and assessed across several quality control metrics includingphysical properties that correlate with circulation viability,immunogenicity, replicative potential, purity, and therapeutic dose. Thecells are then stored in appropriate stabilizing solution and formulatedin a syringe or appropriate delivery vehicle. The cells are infused intothe same patient that donated the initial erythrocytes.

Example 76 Allogeneic Culture Process

To create a scalable, universal therapeutic, etyrhoid cells can becultured from an allogeneic source. The culture of erythroid cells usingallogeneically sourced progenitor CD34+ cells is done to streamline theprocess and culture a volume of therapeutic capable of treating patientsat scale. Donors are blood-typed for major blood antigens, including A,B, Rh to identify universal donors (e.g., O Rh− or Bombay Rh−). CD34+cells from the bone marrow are mobilized to the periphery in a suitabledonor using GM-CSF as described herein. Between 10{circumflex over( )}6-10{circumflex over ( )}8 CD34+ cells are collected and culturedusing the aforementioned 22 day protocol using defined media. During Day4 the cells are transfected with a lentiviral vector containing a genethat codes for the expression of a therapeutic agent. Upon completion ofthe culturing protocol, the cells are purified and assessed acrossseveral quality control metrics including physical properties thatcorrelate with circulation viability, immunogenicity, replicativepotential, purity, and therapeutic dose. The cells are then stored inappropriate stabilizing solution and formulated in a syringe orappropriate delivery vehicle. The cells are then infused into patientsirrespective of their major blood groups.

Example 77 Allogeneic Loading Process

The culture of erythroid cells using allogeneically sourced progenitorCD34+ cells is done to streamline the process to prepare larger volumesof therapeutic cells capable of treating patients at scale. Donors areblood-typed for major blood antigens, including A, B, Rh to identifyuniversal donors (e.g., O Rh− or Bombay Rh−). The cells are loaded witha therapeutic exogenous antigen of interest by one of the methodsdescribed above. Upon completion of the loading protocol, the cells arepurified and assessed across several quality control metrics includingphysical properties that correlate with circulation viability,immunogenicity, replicative potential, purity, and therapeutic dose. Thecells are then stored in appropriate stabilizing solution and formulatedin a syringe or appropriate delivery vehicle. The cells are then infusedinto patients irrespective of their major blood groups.

Example 78 Storage

1. Storage in Refrigerated Buffer Solution

Standard protocols for the storage of red blood cells are known in theart, see e.g., Meryman and Hornblower 1986, Transfusion 26(6):500. Thestandard protocol for the storage of red blood cells (for up to 42 days)is the collection of blood into anticoagulant solutions(citrate-dextrose-phosphate). Erythroid cells are cultured as describedherein. Red cell concentrates are prepared by the removal of plasma bycentrifugation. The cells are stored at 4±2° C. in a slightly hypertonicadditive solution, SAGM (sodium, adenine, glucose, mannitol, 376mOsm/L).

2. Storage in Frozen Buffer Solution

Methods for glycerolization, freezing, and thawing of erythroid cellsare known in the art, see e.g., Meryman and Hornblower 1977 Transfusion17(5):4348. Human blood in citrate phosphate dextrose is glycerolizedand frozen within 4 days of collection. To prepare glycerolized RBCs,approximately 10 mL of whole blood is first centrifuged at 1,400 g for10-15 min, and the plasma is removed. The resulting packed cells arethen glycerolized in two steps using an aqueous glycerol solution withthe following composition: 57.1 g glycerol, 0.03 g potassium chloride,0.085 g magnesium chloride hexahydrate, 0.08 g disodium phosphate, and1.6 g sodium lactate in a total volume of 100 mL, adjusted to a pH of6.8.42 In the first step, 1.5 mL of this glycerol solution is addeddrop-wise to the packed cells with gentle agitation over a period of 3min. The mixture is then allowed to equilibrate undisturbed for at least5 min. In the second glycerolization step, 5 mL of the glycerol solutionis added drop-wise while the mixture is gently agitated over a 3-minperiod, yielding a final glycerol composition of ˜40% w/v. The entireglycerolization process is carried out at room temperature. Theglycerolized RBCs are then divided into aliquots of 0.6-1.1 mL incryogenic vials, placed in a NalgeneVR Cryo “Mr. Frosty” freezingcontainer (Thermo Scientific, N.C.), and stored in a −80 C freezer forat least 12 h and up to 10 years. Frozen RBCs are thawed by placing thecryogenic vial in a 37 C water bath for 1 min. All glycerolized bloodsamples are used in deglycerolization experiments within 2 h of thawing.

3. Formulation as Syringe

The cell population may be intravenously administered via a syringe. Thetherapeutic cells are diluted to a density of 10{circumflex over ( )}7cells/ml using standard saline buffer at 37 C such that 100 ml ofvolume, or 10{circumflex over ( )}9 cells, are delivered. The cellsolution is loaded into a 150 cc syringe, 20 gauge needle and injectedinto the patient through the basilic vein at 5 cc/min. During injectionthe patient's vitals are monitored for any immunogenic or clottingreactions.

4. Formulation as Bag

The cell population may be intravenously administered via syringeconnected to a bag and drip chamber (i.e. an IV drip). The therapeuticcells are diluted to a density of 10{circumflex over ( )}7 cells/mlusing standard saline buffer at 37 C such that 100 ml of volume, or10{circumflex over ( )}9 cells, are delivered. The cell solution isloaded into a 1 L plastic bag, connected to a catheter and allowed todrain via gravity into the patient through the basilic vein. Duringinfusion the patient's vitals are monitored for any immunogenic orclotting reactions.

Example 79 Treatment of Diseases

1. Hemophilia

A patient suffering from hemophilia A is diagnosed. A composition ofexogenous FVIII expressing enucleated hematopoietic cells is prepared asdescribed herein. 10{circumflex over ( )}9 of the cells are administeredintravenously to the patient. The clotting rate is assessed with astandard in vitro clotting time assay known in the art. Circulatingantibodies against FVIII are detected in serum as described herein. Thelevels of circulating antibodies are assessed to track the effectivenessof immune tolerance induction. If the clotting cascade activity isinsufficient to ensure healthy coagulation, recombinant or isolatedFVIII are administered concurrently intravenously in order to reduce thesymptomos of hemophilia A.

2. Atypical Hemolytic Uremic Syndrome

A patient suffering from atypical hemolytic uremic syndrome (aHUS) isdiagnosed. A composition of exogenous CFH expressing enucleatedhematopoietic cells is prepared as described herein. 10{circumflex over( )}9 of the cells are administered intravenously to the patient. Thesymptomatic hemolysis rate is assessed with a standard urinary hemolysisassay known in the art. Circulating antibodies against CFH are detectedin serum as described herein. The levels of circulating antibodies areassessed to track the effectiveness of immune tolerance induction. Thepatient is administered the treatment until the symptoms of the diseaseare seen to ameliorate using the assays described herein.

3. Multiple Sclerosis

An individual with multiple sclerosis (MS) receives a single infusion of1×10{circumflex over ( )}9 antigen expressing enucleated hematopoieticcells expressing the antigenic polypeptide myelin basic protein (MBP),produced and formulated as described herein. At the day of study drugadministration, the patient is monitored in a phase 1 inpatient unit for24 hours. Measurement of the primary outcome is performed at month 3 andadditional safety follow-up is performed until month 6 with consecutiveclinical, MRI, and general physical examinations as well as clinical andlaboratory analyses to assess adverse events and monitor MS diseaseactivity. The procedure is repeated until tolerance is induced such thatthe symptoms of MS are ameliorated in the individual. See, for example,Andreas Lutterotti et al. Sci Transl Med 5, 188ra75 (2013).

Frequency of different cell subsets is analyzed in whole blood (EDTAtubes) by flow cytometry with the following antibody panels: for immunecell subsets (granulocytes, eosinophils, monocytes, and B, T, NK, and NKT cells)—anti-CD45 (PE-Cy7, eBioscience), anti-CD16, (APC-Cy7,BioLegend), anti-CD19 [fluorescein isothiocyanate (FITC), BD], anti-CD14(V450, BD), anti-CD3 [peridinin chlorophyll protein (PerCP), BD], andanti-CD56 [phycoerythrin (PE), eBioscience]; for T cell subsetsincluding CD4+, FoxP3+ Tregs, regulatory CD8+CD57+ILT2+, andproinflammatory CD8+CD161high T cells—anti-CD3 (PE-Cy7, eBioscience),anti-CD4 (APC, eBioscience), anti-CD8 [Pacific Blue (PB), Dako-Biozol],anti-FoxP3 (PE, Miltenyi), anti-CD25 (APC, eBioscience), anti-CD57(FITC, BD), anti-ILT2 (PE, Beckman), and anti-CD161 (APC, Miltenyi). Thecorresponding isotype controls are included in all stainings. Cells areanalyzed with an LSR-II flow cytometer (BD) and FACSDiva Software (BD).

Peripheral blood mononuclear cells (PBMCs) are isolated by Ficolldensity gradient centrifugation (PAA), and functional phenotype of Tcells is evaluated by intracellular cytokine staining as follows:5×10{circumflex over ( )}5 freshly isolated PBMCs are incubatedovernight in 200 ml of X-VIVO 15 (Lonza) in a sterile FACS tube. Thenext day, cells are stimulated with phorbol 12-myristate 13-acetate (50ng/ml, Sigma) and ionomycin (1 mg/ml, Sigma) in the presence ofbrefeldin A (10 mg/ml, eBioscience) for 5 hours. After washing withphosphate-buffered saline, cells are stained with LiveDead kit (AmCyan,Invitrogen), fixed, permeabilized, and stained with differentantibodies: anti-IL-17 (Alexa Fluor 647; eBioscience), anti-IL-4(PE-Cy7, BioLegend), anti-IFN-g (FITC, BioLegend), anti-IL-10 (PE;BioLegend), anti-CD3 (PE, DakoCytomation), anti-CD4 (PB,DakoCytomation), and anti-CD8 (PB, BioLegend) or with the correspondingisotype controls.

The antigen-specific T cell responses toward the myelin peptide used inthe study are measured in freshly isolated PBMCs before the tolerizationprocedure and after 3 months. Antigen-specific T cell responses areanalyzed by proliferation assays with thymidine incorporation. Briefly,isolated PBMCs are seeded in 96-well plates at 1.5×10{circumflex over( )}5 PBMCs per well in X-VIVO 15 medium (Lonza) with 1 mM of peptide.Forty-eight wells are seeded per antigen, and six wells only with mediumas negative control in each plate. TTx (5 mg/ml) (Novartis Behring) isused as positive control. On day 7, plates are incubated for 15 hourswith 1mCi of [3H] thymidine (Hartmann Analytic). [3H] thymidine-pulsedplates are analyzed with a scintillation b counter (Wallac 1450,PerkinElmer). The scintillation counts (CPM) of each well are measured.Wells showing CPM higher than the mean+3 SDs of the unstimulated wellsare considered as positive.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

TABLES A-J

TABLE A Circulating cells Embryonic stem cells (ESC) Induced pluripotentstem cells (iPSC) Cord blood stem cell (CD-SC) Mesenchymal stem cellCD34+ cells Polychromatic normoblasts Hematopoietic stem cells (HSC)Orthochromatic normoblasts Spleen colony forming unit (CFU-S)Proerythroblast Common myeloid progenitor (CMP) Polychromatophilic cellscapable of forming a erythrocyte granulocyte, erythrocyte, monocyte, ormegakaryocyte (CFU-GEMM) Blastocyte colony-forming cells NormoblastBurst-forming unit erythroid (BFU-E) Platelets Megakaryocyte-erythroidprogenitor Leukocytes (MEP) cell Colony-forming unit erythroid (CFU-E)Lymphoid cells Reticulocytes T cells Erythrocytes B cells

TABLE A1 Erythroid cells Embryonic stem cells (ESC) Induced pluripotentstem cells (iPSC) Cord blood stem cell (CD-SC) Polychromatic normoblastsCD34+ cells Orthochromatic normoblasts Hematopoietic stem cells (HSC)Proerythroblast Spleen colony forming unit (CFU-S) Polychromatophilicerythrocyte Common myeloid progenitor (CMP) Normoblast cells capable offorming a granulocyte, erythrocyte, monocyte, or megakaryocyte(CFU-GEMM) Blastocyte colony-forming cells Burst-forming unit erythroid(BFU-E) Megakaryocyte-erythroid progenitor (MEP) cell Erythroid formingcolony unit (CFU-E) Reticulocytes Erythrocytes

TABLE B Circulating cell associated proteins CD1 CD23 CD46 CD72 CD120CD195 CD2 CD24 CD47 CD73 CD122 CD197 CD3 CD25 CD48 CD74 CD127 CD199 CD4CD26 CD49a CD80 CD132 CD209 CD5 CD27 CD49b CD81 CD133 CD202a CD6 CD28CD49c CD82 CD134 CD220 CD7 CD29 CD49d CD83 CD135 CD221 CD8 CD30 CD49eCD86 CD138 CD235a CD9 CD31 CD49f CD87 CD141 CD271 CD10 CD32 CD53 CD88CD142 CD279 CD11a CD33 CD54 CD89 CD143 CD303 CD11b CD34 CD55 CD90 CD144CD304 CD11c CD35 CD56 CD91 CD147 CD309 CD12w CD36 CD57 CD95 CD151 CD326CD13 CD37 CD58 CD96 CD152 TLR 1 CD14 CD38 CD59 CD100 CD154 TLR 2 CD15CD39 CD61 CD103 CD156 TLR 4 CD16 CD40 CD62E CD105 CD158 TLR 5 CD17 CD41CD62L CD106 CD163 TLR 6 CD18 CD42 CD62P CD107 CD165 CD19 CD43 CD63CD107a CD166 CD20 CD44 CD68 CD107b CD168 CD21 CD45 CD69 CD109 CD184 CD22CD71 CD117 CD186

TABLE C Erythrocyte associated proteins 2′,3′-cyclic- Creatine kinaseHypothetical protein RAP1A or RAP1B nucleotide 3′- XP_100510phosphodiesterase Acetylcholinesterase DC 38 Hypothetical protein RAP2BXP_100619 Actin alpha and Duodenal cytochrome Hypothetical protein Rhblood D group beta chain b XP_100665 antigen polypeptide AdenosineEnhancer protein Hypothetical protein Rhesus D category VI deaminaseXP_100925 type III protein Adducin alpha Erythroblast Hypotheticalprotein Similar to adhesive subunit membrane-associated XP_103707 plaquematrix protein protein precursor Aldolase A Far upstream elementHypothetical protein Similar to ankyrin 1 binding protein XP_106269Ankyrin 1 isoform 2 Flotillin 1 Ig heavy chain V-V region Similar toflotillin 2 Ankyrin 1 isoform 4 Flotillin 2 47 Kell Similar toglycophorin A Ankyrin 1 splice Glucose transporter KIAA0340 Similar toLutheran form 2 glycoprotein blood group Aquaporin 1 GlutathioneKIAA1741 protein Similar to RAS-related transferase protein RAB-15Arginase type 1 Glyceraldehyde-3- Lyn B protein Similar to RAS-relatedphosphate protein RAL-A dehydrogenase Arginase type 1 Glycophorin AMembrane protein p55 Similar to tropomyosin erythroid variantATP-binding cassette Glycophorin A Phosphatidylinositol-4- Similar totropomyosin half-transporter precursor phosphate 5 kinase type III 4 18ATP-binding cassette Glycophorin C isoform Phosphoribosyl Solute carrierfamily 2 subfamily C member 1 pyrophosphate synthetase (facilitatedglucose 6 transporter) member 1 bA421H8.2 (novel Hemoglobin alpha Poly(A)-specific Solute carrier family protein) ribonuclease 29 (nucleosidetransporter) member 1 B-CAM protein Hemoglobin betaPresenilin-associated Spectrin alpha chain protein Block of Hemoglobindelta Protein band 3 Spectrin beta chain proliferation 1C-1-tetrahydrofolate Hemoglobin epsilon Protein band 4.1 Translationinitiation synthase factor 2C Calcium transporting Hemoglobin gammaProtein band 4.1 Tropomodulin ATPase 4 (elliptocytosis 1, RH-linked)CD55 HGTD-P Protein band 4.2 Tropomyosin 3 CD58 Hypothetical proteinProtein band 4.9 (dematin) Tropomyosin isoform XP_061743 or XP_089854CD59 antigen Hypothetical protein Protein band 7.2b, TropomyosinalphaXP_091430 stomatin chain (smooth muscle) 26 Cell surface Hypotheticalprotein RAB 35 Unknown protein glycoprotein CD44 XP_091724 Channel-likeintegral Hypothetical protein Rabphilin-3 A-integratingVesicle-associated membrane protein XP_092517 protein membrane protein 2(synaptobrevin 2) Complement Hypothetical protein Ral A binding proteinZona pellucida binding receptor 1 XP_095819 protein Adipocyte plasmaStomatin Myosin-9 Histone H1.1 membrane- associated protein AmmoniumStomatin-like protein Protein 4.1 Histone H2A type 1- transporter Rhtype 2 B/E A Aquaporin 1 Thioredoxin-related Spectrin alpha chain,Histone H3.1 transmembrane erythrocyte protein 4 Aquaporin 7 TMCC2Spectrin beta chain, Histone H4 erythrocyte ATP-binding cassetteTransferrin receptor Talin-1 Lamin A/C sub-family B protein 1 member 6,mitochondrial Band 3 anion Transmembrane and Talin-2 Lamina-associatedtransport protein coiled-coil domain polypeptide 2, isoform family 2alpha Basigin Urea transporter 1 Tropomodulin-1 Lamina-associatedpolypeptide 2, isoforms beta/gamma CD44 Zinc transporter 1 Tropomyosin 1(Alpha) Lamin-B receptor isoform 4 CD47 55 kDa erythrocyte Tropomyosin 3Lamin-B1 membrane protein Equilibrative Actin, alpha cardiac Tropomyosinalpha-3 chain Lamin-B2 nucleoside muscle transporter 1 Erythroid Actin,cytoplasmic Tubulin alpha-1 chain Matrin-3 membrane- associated proteinFlotillin-1 Actin-related protein Tubulin beta chain Multiple inositol 2polyphosphate phosphatase 1 Flotillin-2 Actin-related protein Tubulin,alpha 1 (Testis N-acylneuraminate 2/3 complex subunit specific)cytidylyltransferase 1B Glucose transporter, Actin-related proteinTubulin, alpha 8 Neutral alpha- type 1 2/3 complex subunit 2 glucosidaseAB Glycophorin-A Actin-related protein Tubulin, beta 6 Nuclear porecomplex 3 protein Nup93 Glycophorin-B Alpha-actinin-4 Vinculin Nuclearpore membrane glycoprotein 210 Glycophorin-C Alpha-adducin 78 kDaglucose-regulated Nucleolin protein Immunoglobulin-like Ankyrin-1Antigen KI-67 Nucleoporin NUP188 domain-containing homolog receptor 1Integrin alpha-X Ankyrin-3 ATP-dependent RNA Nucleoprotein TPR helicaseDDX39A Integrin beta-1 Beta-actin-like protein Calnexin Prelamin-A/C 2Kell blood group Beta-adducin Calreticulin Protein disulfide-glycoprotein isomerase Large neutral amino Capping protein (Actin DNAtopoisomerase 1 Protein disulfide- acids transporter filament) muscle Z-isomerase A4 small subunit 3 line, beta Membrane Cortactin DNA-dependentprotein Protein disulfide- transport protein XK kinase catalytic subunitisomerase A6 Membrane- Dematin Dolichyl- Protein ERGIC-53 associateddiphosphooligosaccharide- progesterone protein glycosyltransferasereceptor component 48 kDa subunit 2 Monocarboxylate Dynactin 2 (P50),Dolichyl- Ribophorin II transporter 1 isoform CRA_bdiphosphooligosaccharide- protein glycosyltransferase subunit 1Multidrug Erythrocyte Endoplasmic reticulum Transitional resistance-membrane protein resident protein 29 endoplasmic reticulum associatedprotein 4 band 4.2 ATPase Neutral cholesterol Filamin-A Endoplasmicreticulum UDP- ester hydrolase 1 resident protein 44glucose:glycoprotein glucosyltransferase 1 Plasma membrane Gamma-adducinEndoplasmin CD59 calcium-transporting ATPase 1 Plasma membrane GelsolinER-Golgi SNARE of 24 kDa calcium-transporting ATPase 3 Plasma membraneKinesin-1 heavy chain FACT complex subunit calcium-transporting SPT16ATPase 4 Probable E3 Microtubule- Glucosidase 2 subunit betaubiquitin-protein associated protein ligase C12orf51 RP/EB family member1 Rh blood group, Myosin light chain 4 Heme oxygenase 1 CcEe antigensSLC43A3 Myosin light Hemogen polypeptide 6 Sodium/calcium Myosin, heavychain Heterochromatin protein exchanger SCL8A3 11, smooth muscle1-binding protein 3 Sodium/potassium- Myosin-10 High mobility grouptransporting ATPase protein B1 subunit alpha-1 Sodium/potassium-Myosin-14 High mobility group transporting ATPase protein B2 subunitbeta-3

TABLE C1 Erythrocyte transmembrane proteins Aquaporin 1 Kell Cellsurface glycoprotein CD44 Membrane protein p55 Channel-like integralProtein band 3 membrane protein Complement receptor 1 Rh blood D groupantigen polypeptide Erythroblast membrane- Rhesus D category VI type IIIprotein associated protein Glucose transporter glycoprotein Similar toglycophorin A Glycophorin A Similar to Lutheran blood group GlycophorinA precursor Solute carrier family 2 (facilitated glucose transporter)member 1 Glycophorin C isoform 1 Solute carrier family 29 (nucleosidetransporter) member 1

TABLE C2 Erythrocyte GPI-linked proteins Acetylcholinesterase CD55 CD58CD59 antigen

TABLE C3 Erythrocyte intracellular proteins 2′,3′-cyclic-nucleotide 3′-Enhancer protein Hypothetical protein RAP1A or RAP1B phosphodiesteraseXP_100925 Actin alpha and beta Far upstream element Hypothetical proteinRAP2B chains binding protein XP_103707 Adenosine deaminase Flotillin 1Hypothetical protein Similar to adhesive XP_106269 plaque matrix proteinprecursor Adducin alpha subunit Flotillin 2 47 Ig heavy chain V-VSimilar to ankyrin 1 region Aldolase A Glutathione transferase KIAA0340Similar to flotillin 2 Ankyrin 1 isoform 2 Glyceraldehyde-3- KIAA1741protein Similar to RAS-related phosphate dehydrogenase protein RAB-15Ankyrin 1 isoform 4 Hemoglobin alpha Lyn B protein Similar toRAS-related protein RAL-A Ankyrin 1 splice form 2 Hemoglobin betaPhosphatidylinositol-4- Similar to tropomyosin phosphate 5 kinase typeIII Arginase type 1 Hemoglobin delta Phosphoribosyl Similar totropomyosin 4 pyrophosphate synthetase Arginase type 1 erythroidHemoglobin epsilon Poly (A)-specific Spectrin alpha chain variantribonuclease ATP-binding cassette Hemoglobin gamma Presenilin-associatedSpectrin beta chain half-transporter protein ATP-binding cassette HGTD-PProtein band 4.1 Translation initiation subfamily C member 6 factor 2CbA421H8.2 (novel Hypothetical protein Protein band 4.1 Tropomodulinprotein) XP_061743 or XP_089854 (elliptocytosis 1, RH- linked) B-CAMprotein Hypothetical protein Protein band 4.2 Tropomyosin 3 XP_091430Block of proliferation 1 Hypothetical protein Protein band 4.9Tropomyosin isoform XP_091724 (dematin) C-1-tetrahydrofolateHypothetical protein Protein band 7.2b, Tropomyosinalpha chain synthaseXP_092517 stomatin (smooth muscle) 26 Calcium transporting Hypotheticalprotein RAB 35 Unknown protein ATPase 4 XP_095819 Creatine kinaseHypothetical protein Rabphilin-3 A- Vesicle-associated XP_100510integrating protein membrane protein 2 (synaptobrevin 2) DC 38Hypothetical protein Ral A binding protein Zona pellucida bindingXP_100619 protein Duodenal cytochrome b Hypothetical protein XP_100665

TABLE D Conjugation methods Zero-length x-linker EDC EDC plus sulfo NHSCMC DCC DIC Woodward's reagent K N,N′-carbonyldiimidazole Schiff base +reductive amination Homobifunctional NHS esters DSP DTSSP DSSBS{circumflex over ( )}3 DST Sulfo-DST BSOCOES Sulfo-BSOCOES EGSSulfo-EGS DSG DSC Homobifunctional Imidoesters DMA DMP DMS DTBPSulfhydryl reactive x-linkers DPDPB BMH Difluorobenzene derivativesDFDNB DFDNPS Photoreactive x-linker BASED Homobifunctional aldehydesFormaldehyde Glutaraldehyde bis-epoxide 1,4-butanediol diglycidyl etherHomobifunctional hydrazides adipic acid dihydrazide carbohydrazideBis-diazonium derivative o-tolidine diazotized Bis-diazotized benzidineAmine-sulfhydryl x-linker SPDP, LC-SPDP, sulfo-LC-SPDP SMPT andsulfo-LC-SMPT SMCC and sulfo-SMCC MBS and sulfo-MBS SIAB and sulfo-SIABSMPB and sulfo-SMPB GMBS and sulfo-GMBS SIAX and SIAXX SIAC and SIACXNPIA Carbonyl-sulfydryl x-linker MPBH M2C2H PDPH amine-photoreactivex-linker NHS-ASA, Sulfo-NHS-ASA Sulfo-NHS-LC-ASA SASD HSAB andsulfo-HSAB SANPAH and sulfo-SANPAH ANB-NOS SAND SADP and sulfo-SADPSulfo-SAPB SAED Sulfo-SAMCA p-Nitrophenyl diazopyruvate PNP-DTPsulfhydryl-photoreactive x- linker ASIB APDPBenzophenone-4-iodoacetamide Benzophenone-4-maleimideCarbonyl-photoreactive x-linker ABH Carboxylate-photoreactive x- linkerASBA arginine-photoreactive x-linker APG Bioorthogonal reactionsDiels-alder reagent pairs Hydrazine-aldehyde reagent pairs Boronic acidsalicylhydroxamate Click chemistry Staudinger ligation

TABLE D1 Enzymatic conjugation methods Enzymatic reactionsSpyCatcher/SpyTag Spy0128 derivatives Transpeptidases IsopeptidasesSortase DD-transpeptidase Peptidyl transferase G-glutamyl transpeptidaseD-glutamyl transpeptidase Farnesyltransferase PrenyltranferaseDimethylallyltrans-transferase Geranylgeranyl pyrophosphate synthaseDehydrodolichol diphosphate synthase

TABLE E Chemistry of reactive groups Amine reactions IsothyocyantesIsocyanates Acyl azides NHS esters Sulfonyl chlorides Aldehydes andglyoxals Epoxides and oxiranes Carbonates Arylating agents ImidoestersCarbodiimides Anhydrides Fuorphenyl esters Hydroxymethyl phosphinederivatives Guanidination of amines Thiol reactions Haloacetyl and alklhalide derivatives Maleimides Aziridines Acryloyl derivatives Arylatingagents Thil-disulfide exchange reagents Vinylsulfone derivativesMetal-thiol dative bonds Carboxylate rections Diazoalkanes anddiazoacetyl compounds Carbonyldiimidazole Carbodiimides Hydroxylreactions Epoxides and oxiranes Carbonyldiimidazole N,N′0disuccinimidylcarbonate N-hydroxysuccinimidyl chloroformate Oxidation with periodateEnzymatic oxidation Alkyl halogens Isocyanates Aldehyde and Ketonereactions Hydrazine derivatives Schiff base formation Reductiveamination Mannich condensation Active hydrogen reactions Diazoniumderivatives Mannich condensation Iodination reactions Cycloadditionreactions Diels-Alder reaction Complex formation with boronic acidderivatives Click chemistry: Cu- promoted Azide- Alkyne [3 + 2]cycloaddition

TABLE F Autoimmune diseases and antigens Disease Known antigen Acuterheumatic fever cross reactive antibodies to cardiac muscle alopeciaareata Trychohyalin, keratin 16 ANCA-associated vasculitis Neutrophilcytoplasmic antigen, proteinase 3, myeloperodixase, bacterialpermiability increasing factor autoimmune gastritis H, K adenosinetriphosphatase autoimmune hemolytic Rh blood group antigens, I antigenanemia autoimmune hepatitis nuclear protein, liver-kidney microsome type1, liver cytosol type 1 autoimmune myocarditis cardiac myosin Autoimmunethyroiditis Thyroid peroxidase, thyroglobulin, thyroid- stimulatinghormone receptor Autoimmune uveitis Retinal arrestin (S-antigen)dermatomyositis Mi2 ATPase diabetes (type 1) Pancreatic beta cellantigen goodpasture's syndrome Noncollagenous domain of basementmembrane collagen type IV Graves' disease Thyroid stimulating hormonereceptor Guillain-Barre syndrome Neurofascin-186, gliomedin, nodaladhesion molecueles Hypoglycemia Insulin receptor idiopathic Plateletintegrin GpIIb, GpIIIa thrombocytopenic purpura Insulin resistantdiabetes Insulin receptor Membranous nephritis Phospholipase A2 mixedessential rheumatoid factor IgG complexes cryoglobulinemia multiplesclerosis Myelin basic protein, proteolipid protein, myelinoligodendrocyte glycoprotein myasthenia gravis Acetylcholine receptorMyasthenia gravis - MUSC Muscarinic receptor pemphigus/pemphigoidEpidermal cadherin pernicious anemia intrinsic factor (Gastric)polymyositis nuclear and nucleolar antigen primary biliary cirrhosisneutrophil nuclear antigen, mitochondrial multienzyme complex psoriasisPSO p27 rheumatoid arthritis rheumatoid factor IgG complexes, synovialjoint antigen, citrullinated protein, carbamylated proteinscleroderma/systemic Scl-86, nucleolar scleroderma antigen sclerosisSjogren's syndrome SS-B, Lupus La protein systemic lupus DNA, histones,ribosomes, snRNP, scRNP erythematosus vitiligo VIT-90, VIT-75, VIT-40Wegener's granulomatosis neutrophil nuclear antigen AntiphospholipidBeta-2 glycoprotein 1 syndrome (APS) & catastrophic APS Chemotherapyinduced Neuronal antigens peripheral neuropathy Thrombotic ADAMTS13thrombocytopenic purpura Atypical hemolytic uremic Complement factor Hsyndrome

TABLE G Inflammatory diseases and antigens Disease Antigen Crohn'sdisease Flagellin, microbial antigens Ulcerative colitis Neutrophilcytoplasmic antigen, microbial antigens Celiac disease GlutenInflammatory bowel disease Microbial antigens

TABLE H Allergic disease triggers Allergy Antigen Animal fel d 1, can f6dander Black 2S albumin, vicilin-like (7S) protein walnut Brazil 2Salbumin, legumin-like (11S) seed storage protein nut Cashew 7Svicilin-like protein, legumin-like 11S seed storage protein nut ChestnutChitinase 1b, lipid transfer protein Cas s8 Dust Der p2 mites EggOvomucoid, ovalbumin, ovotransferrin, lysozyme, alpha-livetin English 2Salbumin, 7S vicilin-like protein, lipid transfer protein, legumin-like11S seed storage walnut protein Fish Parvalbumins Hazelnut Bet v1homologue, profilin, lipid transfer protein, 11s globulin-like protein,7S vicilin-like protein Insect Melittin, phospholipase A2,hyaluronidase, acid phosphatase, protease, antigen 5, Api venom m1-4,Bom p1, p4, Dol m1, 2,5; Vesp c1, c5; Pol a1, a2, a5; Ves v1, v2, v5;Sol|1-4 Latex Hev b 1, 2, 3, 5, 6.01, 6.02, 8, 9, 11 Milk Alpha s1casein, beta-lactoglobulin Mold enzymes, toxins, cell wall componentsPeanut Ara h1, Ara h2, Ara h3, Ara h6 Pollen Aconitate hydratase,fructose bisphosphate aldolase, ATP synthase, luminal binding protein,calmodulin, calreticulin, chaperonin, enolase, lipid transfer protein 1,lipid transfer protein 2, profilins Pollen, Phl p 1, 2, 4, 5, 6, 11, 12,13 grass Shellfish arginine kinase, tropomyosin, myosin light chain,sarcoplasmic calcium binding protein, triose phosphate isomerase,aldolase, titin Soy Gly m1 soybean hydrophobic protein, gly m4, gly m5,gly m6, Gly m 2s albumin, lipid transfer proteins, alpha-globulin, TreeLipid transfer proteins, profilins, Bet v1-related family, legumins,vicilins, 2S albumins nuts Wheat gluten, prolamins, 2S albumins, lipidtranser proteins, a-amylase/protease inhibitors, puroindoline,alpha-globulin, alpha-gliadin, beta-gliadin, gamma-gliadin, fast-omega-gliadin, slow-omega-gliadin

TABLE I Therapeutic proteins to treat diseases Brand Name CompanyIndication Amevive Astellas Pharma Moderate to severe chronic plaquepsoriasis BayGam Bayer Hepatitis A, measles, varicella, rubella,immunoglobulin deficiency CinnoVex CinnaGen Multiple Sclerosis SynagisMedlummne Respiratory syncytial virus (RSV) infections Lucentis RocheGenentech Wet age-related macular degeneration (AMD) Actemra Hoffman-LaRoche Rheumatoid Arthritis Avastin Roche Genentech Various cancersBenefix Pfizer Heamophelia B/Christmas disease (coagulation factor IXdefficiency) Benlysta HGS, GlaxoSmithKline Systemic lupus erythematosus(SLE) Bexxar GlaxoSmithKline, Corixa CD20 +. follicular, NHL CampathGenzyme B-cell chronic lymphocytic leukemia (B-CLL Ceredase Genzyme TypeI Gaucher disease Cerezyme Genzyme Type 1 Gaucher's disease ErbituxLilly Metastatic colorectal cancer Helixate FS CSL Behring Haemophilia AHerceptin Roche Genentech Breast cancer Kogenate FS Bayer HealthcareHaemophilia A Lumizyme Genzyme Pompe disease (glycogen storage diseasetype II) NovoSeven Novo Nordisk Hemophilia A or B patients withinhibitors to Factor VIII or Factor IX and in patients with acquiredhemophilia Privigen CSL Behring IVIG therapy Recombinate Baxter, WyethHaemophilia A Refacto Pfizer Haemophilia A Remicade Jannssen Biotech(J&J) Rheumatoid Arthritis, Crohn's Disease, Ankylosing Spondylitis,Plaque Psoriasis, Ulcerative Colitis ReoPro Lilly PTCA (Angioplasty)adjunct Rituxan/MabThera Roche Genentech/Biogen Blood cancers andrheumatoid arthritis Idec Simulect Novartis Organ rejection prophylaxisSoliris Alexion Pharmaceuticals Paroxysmal nocturnal hemoglobinuria(PNH), atypical hemolytic uremic syndrome (aHUS) Tysabri Biogen Idec,Elan Pharma. Multiple Sclerosis, Crohn's Disease Vectibix AmgenTreatment of epidermal growth factor receptor (EGFR)-expressing,metastatic colorectal carcinoma (mCRC) Xyntha Wyeth Haemophilia A(factor VIII defficiency) Zenapax Roche/PDL Prophylaxis of acute organrejection in patients receiving renal transplants Arcalyst RegeneronPharmaceuticals Cryopyrin-Associated Periodic Syndromes (CAPS),including FCAS and MWS Betaseron Bayer Healthcare Multiple Sclerosis,myocardial disease Cetrotide Merck Serono Infertility Cimzia UCBRheumatoid Arthritis, Crohn's Disease Copaxone Teva PharmaceuticalsMultiple Sclerosis, Crohn's disease, glaucoma, motor neurone disease,Huntingtons chorea, neurodegenerative disease Enbrel Amgen, WyethRheumatoid Arthritis, psoriasis Epogen Amgen Anemia Humira AbbottRheumatoid Arthritis, Crohn's Disease Kineret Amgen, Biovitrum ActiveRheumatoid Arthritis Lantus Sanofi-Aventis Diabetes Pegasys RocheGenentech Chronic hepatitis C, chronic hepatitis B Prolia Amgen, GSKOsteoporosis, therapy-induced bone loss (breast or ovarian cancer), bonemetastatase, giant cell tumor of bone, multiple myeloma — — — RebifMerck Serono, Pfizer Multiple Sclerosis (relapsing) Simponi Johnson &Johnson Rheumatoid Arthritis, Crohn's Disease, Ulcerative Cilitis,Ankylosing Spondylitis, Psoriatic Arthritis Stelara Centocor,Janssen-Cilag Moderate to severe plaque psoriasis who are candidates forphototherapy or systemic therapy. Vivaglobin CSL Behring Primary ImmuneDefficiency (PID) Xgeva Amgen Bone Metastasis from Solid Tumors XolairRoche Genentech, Novartis Moderate to severe persistent allergic asthmaAvonex Biogen Idec Multiple Sclerosis (relapsing) Aranesp Amgen AnemiaOrencia Bristol-Myers-Squibb Rheumatoid arthritis, juvenile ideopathicarthritis Procrit Jannssen Biotech (J&J) Anemia Erwinaze Jazz Acutelymphoblastic luekemia

TABLE J Therapeutic protein classes to treat diseases AAV capsid proteinAsparaginase alglucosidase alpha CTLA4 Anti C5 Erythropoietin Anti gplib/IIIa Factor IX Anti IGE Factor VII Anti IL-12, Anti IL-23 FactorVIII Anti RANK ligand Glatiramer acetate Anti-alpha 4 integringlucocerebrosidase Anti-APRIL GnRH antagonist Anti-BAFF IgG Anti-CD20IL1R antagonist Anti-CD52 IL1R or IL1 antagonist Anti-EGFR InsulinAnti-Her2 Interferon alpha Anti-IL2 receptor interferon beta Anti-IL6receptor Lentivirus capsid protein Anti-PD1 LFA3-Fc Anti-RSV protein FRetrovirus capsid protein Anti-TNFa TACI-Ig Anti-VEGF TNF-receptor

TABLES 1-8

TABLE 1 Erythroid Polypeptides and Non-Exogenous antigen PolypeptidesABO blood groups Stomatin Peters DAF Cromer Aquaporin 3 TropomyosinRasmussen Gerbich (GYPC) Aubergers Glucose transporter Reid CD47 Band 3Adducin REIT Glycophorin A, B, C Basigin Rabphilin SARA Band 3 (AE3) C41C1 tetrahydrofolate Rhesus blood D group GYPB Ss synthase CD44 Vel groupAldolase C4A, C4B Chido, Rodgers C4 component of complement Cis AB Lanantigen Tropomodulin HLA Bg HLA class I Diego (Di) At antigen ArginaseRHAG Rh-associated Ammonium transport Colton antigen Jr antigen Creatinekinase glycoprotein Complement Component 4 AnWj antigen B-Cam proteinColton (Co) Water channel protein alpha(1,3) Sd antigen Rap1A ACHECartwright (Yt) fucosyltransferase Acetylcholinesterase CR1 BattyBennett-Goodspeed Glutathione transferase DAF Bilkes P antigen systemGlycophorin C Diego Wright (Wr) Rh blood group Aquaporin Duffy Box Xgantigen system Erythroblast associated membrane protein Hh/Bombayantigen Christiansen XK protein CD44 ii antigen alpha(1,2) Yt/Cartwrightantigen Synaptobrevin 2 fucosyltransferase system Indian blood group HJKCD58 Ribonuclease Kell HOFM Rh ABO glycosyl transferases Kidd JFV AnWjAdhesion receptor CD59 Lewis antigen JONEs Scianna CD44 Lutheran antigenJensen Radin MER2 MNS antigen system Katagiri Duodenal cytochrome B DOKDombrock ADP- ribosyltransferase Cost group Livesay DARC (Duffy) SEMA7AJMH Putative adhesion receptor Er group Milne CR1 Knops-McCoy UMOD SdaTamm- Horsfall protein (uromodulin) Dematin Oldeide FP Family Anionexchanger channel protein (band 3, AE1) Indian (In) Annexin FamilyTweety Family CTL Family Kidd (Jk) Urea Bcl-2 Family UT Family DAACSFamily transporter FUT3 Lewis (Le) Bestrophin Family VIC Family DASSfamily Adenosine deaminase BNip3 Family AAAP Family DMT family OK OkaNeurothelin, CD20 Family transferrin receptor ENT Family putativeadhesion molecule LW Adhesion receptor CLIC Family c-KIT GPH Family FUT2Secretor (Se) Connexin Family Insulin receptors 1 & 2 GUP Family FUT1 Hhalpha CRAC-C Family Estrogen receptor LCT Family LU Lutheran (Lu) CtrFamily Dexamethasone receptor MC family Adhesion receptor P1Glycosyltransferase E-CIC Family JAK2 kinase MET Family XK Kx PutativeENaC Family ABC family MFS Family neurotransmitter transporter XG Xgformerly called GIC Family ArsAB family MOP Family PBDX MIC2 ICC FamilyF-ATPase Family MTC Family Hemoglobin Innexin Family IISP Family NCS2Family Ankyrin IRK-C Family MPT Family Nramp Family Spectrin LIC FamilyP-ATPase Family NSS Family KEL Kell (K, k, Kp, Js) MIP Family AE familyOAT Family Metalloproteinase Torkildsen MIT family APC Family OST FamilyRab 35 NSCC2 Family ArsB Family Oxa1 Family Ral A binding protein PCCFamily BASS Family PiT Family Zona pellucida binding Plamolipin FamilyCaCA Family PNaS Family protein Lyn B protein PLB Family CCC Family POTFamily Klaa1741 protein PLM Family CDF Family RFC Family DC38 PresenilinFamily CIC Family RND Family* Calciums transporting RIR-CaC Family CNTFamily SSS Family ATPase ACC Family TRIC Family CPA1 Family STRA6 FamilyAmt Family TRP-CC Family CPA2 Family SulP Family ZIP Family HCC FamilyNIPA Family N-MDE Family ATP-E Family LPI Family PPI Family Epo receptordsRNA-T Family MagT1 Family PPI2 Family MgtE Family

TABLE 2 Erythroid Promoters Promoter Gene beta globin promoter betaglobin 3′ beta-globin enhancer beta globin beta globin locus controlregion beta globin GATA-1 promoter GATA-1 GYPA promoter Glycophorin AHK1 promoter Hexokinase

TABLE 3 Sequences of Complement Receptor 14A. CR1 isoform S precursor, Homo sapiens NCBI Reference Sequence No. NP_000642.31 mgassprspe pvgppapglp fccggsllav vvllalpvaw gqcnapewlp farptnltde 61fefpigtyln yecrpgysgr pfsiiclkns vwtgakdrcr rkscrnppdp vngmvhvikg 121iqfgsqikys ctkgyrligs ssatciisgd tviwdnetpi cdripcglpp titngdfist 181nrenfhygsv vtyrcnpgsg grkvfelvge psiyctsndd qvgiwsgpap qciipnkctp 241pnvengilvs dnrslfslne vvefrcqpgf vmkgprrvkc qalnkwepel pscsrvcqpp 301pdvlhaertq rdkdnfspgq evfyscepgy dlrgaasmrc tpqgdwspaa ptcevkscdd 361fmgqllngrv lfpvnlqlga kvdfvcdegf qlkgssasyc vlagmeslwn ssvpvceqif 421cpsppvipng rhtgkplevf pfgktvnytc dphpdrgtsf dligestirc tsdpqgngvw 481sspaprcgil ghcqapdhfl faklktqtna sdfpigtslk yecrpeyygr pfsitcldnl 541vwsspkdvck rkscktppdp vngmvhvitd iqvgsrinys cttghrligh ssaecilsgn 601aahwstkppi cqripcglpp tiangdfist nrenfhygsv vtyrcnpgsg grkvfelvge 661psiyctsndd qvgiwsgpap qciipnkctp pnvengilvs dnrslfslne vvefrcqpgf 721vmkgprrvkc qalnkwepel pscsrvcqpp pdvlhaertq rdkdnfspgq evfyscepgy 781dlrgaasmrc tpqgdwspaa ptcevkscdd fmgqllngrv lfpvnlqlga kvdfvcdegf 841qlkgssasyc vlagmeslwn ssvpvceqif cpsppvipng rhtgkplevf pfgktvnytc 901dphpdrgtsf dligestirc tsdpqgngvw sspaprcgil ghcqapdhfl faklktqtna 961sdfpigtslk yecrpeyygr pfsitcldnl vwsspkdvck rkscktppdp vngmvhvitd 1021iqvgsrinys cttghrligh ssaecilsgn aahwstkppi cqripcglpp tiangdfist 1081nrenfhygsv vtyrcnpgsg grkvfelvge psiyctsndd qvgiwsgpap qciipnkctp 1141pnvengilvs dnrslfslne vvefrcqpgf vmkgprrvkc qalnkwepel pscsrvcqpp 1201pdvlhaertq rdkdnfspgq evfyscepgy dlrgaasmrc tpqgdwspaa ptcevkscdd 1261fmgqllngrv lfpvnlqlga kvdfvcdegf qlkgssasyc vlagmeslwn ssvpvceqif 1321cpsppvipng rhtgkplevf pfgkavnytc dphpdrgtsf dligestirc tsdpqgngvw 1381sspaprcgil ghcqapdhfl faklktqtna sdfpigtslk yecrpeyygr pfsitcldnl 1441vwsspkdvck rkscktppdp vngmvhvitd iqvgsrinys cttghrligh ssaecilsgn 1501tahwstkppi cgripcglpp tiangdfist nrenfhygsv vtyrcnlgsr grkvfelvge 1561psiyctsndd qvgiwsgpap qciipnkctp pnvengilvs dnrslfslne vvefrcqpgf 1621vmkgprrvkc qalnkwepel pscsrvcqpp peilhgehtp shqdnfspgq evfyscepgy 1681dlrgaaslhc tpqgdwspea prcavkscdd flgqlphgrv lfplnlqlga kvsfvcdegf 1741rlkgssvshc vlvgmrslwn nsvpvcehif cpnppailng rhtgtpsgdi pygkeisytc 1801dphpdrgmtf nligestirc tsdphgngvw sspaprcels vraghcktpe qfpfasptip 1861indfefpvgt slnyecrpgy fgkmfsiscl enlvwssved ncrrkscgpp pepfngmvhi 1921ntdtqfgstv nyscnegfrl igspsttclv sgnnvtwdkk apiceiisce ppptisngdf 1981ysnnrtsfhn gtvvtyqcht gpdgeqlfel vgersiycts kddqvgvwss ppprcistnk 2041ctapevenai rvpgnrsfft lteiirfrcq pgfvmvgsht vqcqtngrwg pklphcsrvc 2101qpppeilhge htlshqdnfs pgqevfysce psydlrgaas lhctpqgdws peaprctvks 2161cddflgqlph grvllplnlq lgakvsfvcd egfrlkgrsa shcvlagmka lwnssvpvce 2221qifcpnppai lngrhtgtpf gdipygkeis yacdthpdrg mtfnligess irctsdpqgn 2281gvwsspaprc elsvpaacph ppkiqnghyi gghvslylpg mtisyicdpg yllvgkgfif 2341ctdqgiwsql dhyckevncs fplfmngisk elemkkvyhy gdyvtlkced gytlegspws 2401qcqaddrwdp plakctsrth dalivgtlsg tiffilliif lswiilkhrk gnnahenpke 2461vaihlhsqgg ssvhprtlqt neensrvlp (Seq. ID No. 1)4B. CR1 isoform F precursor, Homo sapiens NCBI Reference Sequence No. NP_000564.21 mgassprspe pvgppapglp fccggsllav vvllalpvaw gqcnapewlp farptnltde 61fefpigtyln yecrpgysgr pfsiiclkns vwtgakdrcr rkscrnppdp vngmvhvikg 121iqfgsqikys ctkgyrligs ssatciisgd tviwdnetpi cdripcglpp titngdfist 181nrenfhygsv vtyrcnpgsg grkvfelvge psiyctsndd qvgiwsgpap qciipnkctp 241pnvengilvs dnrslfslne vvefrcqpgf vmkgprrvkc qalnkwepel pscsrvcqpp 301pdvlhaertq rdkdnfspgq evfyscepgy dlrgaasmrc tpqgdwspaa ptcevkscdd 361fmgqllngrv lfpvnlqlga kvdfvcdegf qlkgssasyc vlagmeslwn ssvpvceqif 421cpsppvipng rhtgkplevf pfgktvnytc dphpdrgtsf dligestirc tsdpqgngvw 481sspaprcgil ghcqapdhfl faklktqtna sdfpigtslk yecrpeyygr pfsitcldnl 541vwsspkdvck rkscktppdp vngmvhvitd iqvgsrinys cttghrligh ssaecilsgn 601aahwstkppi cqripcglpp tiangdfist nrenfhygsv vtyrcnpgsg grkvfelvge 661psiyctsndd qvgiwsgpap qciipnkctp pnvengilvs dnrslfslne vvefrcqpgf 721vmkgprrvkc qalnkwepel pscsrvcqpp pdvlhaertq rdkdnfspgq evfyscepgy 781dlrgaasmrc tpqgdwspaa ptcevkscdd fmgqllngrv lfpvnlqlga kvdfvcdegf 841qlkgssasyc vlagmeslwn ssvpvceqif cpsppvipng rhtgkplevf pfgkavnytc 901dphpdrgtsf dligestirc tsdpqgngvw sspaprcgil ghcqapdhfl faklktqtna 961sdfpigtslk yecrpeyygr pfsitcldnl vwsspkdvck rkscktppdp vngmvhvitd 1021iqvgsrinys cttghrligh ssaecilsgn tahwstkppi cgripcglpp tiangdfist 1081nrenfhygsv vtyrcnlgsr grkvfelvge psiyctsndd qvgiwsgpap qciipnkctp 1141pnvengilvs dnrslfslne vvefrcqpgf vmkgprrvkc qalnkwepel pscsrvcqpp 1201peilhgehtp shqdnfspgq evfyscepgy dlrgaaslhc tpqgdwspea prcavkscdd 1261flgqlphgrv lfplnlqlga kvsfvcdegf rlkgssvshc vlvgmrslwn nsvpvcehif 1321cpnppailng rhtgtpsgdi pygkeisytc dphpdrgmtf nligestirc tsdphgngvw 1381sspaprcels vraghcktpe qfpfasptip indfefpvgt slnyecrpgy fgkmfsiscl 1441enlvwssved ncrrkscgpp pepfngmvhi ntdtqfgstv nyscnegfrl igspsttclv 1501sgnnvtwdkk apiceiisce ppptisngdf ysnnrtsfhn gtvvtyqcht gpdgeqlfel 1561vgersiycts kddqvgvwss ppprcistnk ctapevenai rvpgnrsfft lteiirfrcq 1621pgfvmvgsht vqcqtngrwg pklphcsrvc qpppeilhge htlshqdnfs pgqevfysce 1681psydlrgaas lhctpqgdws peaprctvks cddflgqlph grvllplnlq lgakvsfvcd 1741egfrlkgrsa shcvlagmka lwnssvpvce qifcpnppai lngrhtgtpf gdipygkeis 1801yacdthpdrg mtfnligess irctsdpqgn gvwsspaprc elsvpaacph ppkiqnghyi 1861gghvslylpg mtisyicdpg yllvgkgfif ctdqgiwsql dhyckevncs fplfmngisk 1921elemkkvyhy gdyvtlkced gytlegspws qcqaddrwdp plakctsrth dalivgtlsg 1981tiffilliif lswiilkhrk gnnahenpke vaihlhsqgg ssvhprtlqt neensrvlp(Seq. ID No. 2)4C. Predicted CR1 isoform X1, Homo sapiens, NCBI Reference Sequence No. XP_005273121.11 mclgrmgass prspepvgpp apglpfccgg sllavvvlla lpvawgqcna pewlpfarpt 61nltdefefpi gtylnyecrp gysgrpfsii clknsvwtga kdrcrrkscr nppdpvngmv 121hvikgiqfgs qikysctkgy rligsssatc iisgdtviwd netpicdrip cglpptitng 181dfistnrenf hygsvvtyrc npgsggrkvf elvgepsiyc tsnddqvgiw sgpapqciip 241nkctppnven gilvsdnrsl fslnevvefr cqpgfvmkgp rrvkcqalnk wepelpscsr 301vcqpppdvlh aertqrdkdn fspgqevfys cepgydlrga asmrctpqgd wspaaptcev 361kscddfmgql lngrvlfpvn lqlgakvdfv cdegfqlkgs sasycvlagm eslwnssvpv 421ceqifcpspp vipngrhtgk plevfpfgkt vnytcdphpd rgtsfdlige stirctsdpq 481gngvwsspap rcgilghcqa pdhflfaklk tqtnasdfpi gtslkyecrp eyygrpfsit 541cldnlvwssp kdvckrksck tppdpvngmv hvitdiqvgs rinyscttgh rlighssaec 601ilsgnaahws tkppicqrip cglpptiang dfistnrenf hygsvvtyrc npgsggrkvf 661elvgepsiyc tsnddqvgiw sgpapqciip nkctppnven gilvsdnrsl fslnevvefr 721cqpgfvmkgp rrvkcqalnk wepelpscsr vcqpppdvlh aertqrdkdn fspgqevfys 781cepgydlrga asmrctpqgd wspaaptcev kscddfmgql lngrvlfpvn lqlgakvdfv 841cdegfqlkgs sasycvragm eslwnssvpv ceqifcpspp vipngrhtgk plevfpfgkt 901vnytcdphpd rgtsfdlige stirctsdpq gngvwsspap rcgilghcqa pdhflfaklk 961tqtnasdfpi gtslkyecrp eyygrpfsit cldnlvwssp kdvckrksck tppdpvngmv 1021hvitdiqvgs rinyscttgh rlighssaec ilsgnaahws tkppicqlcq pppdvlhaer 1081tqrdkdnfsp gqevfyscep gydlrgaasm rctpqgdwsp aaptcevksc ddfmgqllng 1141rvlfpvnlql gakvdfvcde gfqlkgssas ycvlagmesl wnssvpvceq ifcpsppvip 1201ngrhtgkple vfpfgkavny tcdphpdrgt sfdligesti rctsdpqgng vwsspaprcg 1261ilghcqapdh flfaklktqt nasdfpigts lkyecrpeyy grpfsitcld nlvwsspkdv 1321ckrkscktpp dpvngmvhvi tdiqvgsrin yscttghrli ghssaecils gntahwstkp 1381picgripcgl pptiangdfi stnrenfhyg svvtyrcnlg srgrkvfelv gepsiyctsn 1441ddqvgiwsgp apqciipnkc tppnvengil vsdnrslfsl nevvefrcqp gfvmkgprrv 1501kcqalnkwep elpscsrvcq pppeilhgeh tpshqdnfsp gqevfyscep gydlrgaasl 1561hctpqgdwsp eaprcavksc ddflgqlphg rvlfplnlql gakvsfvcde gfrlkgssvs 1621hcvlvgmrsl wnnsvpvceh ifcpnppail ngrhtgtpsg dipygkeisy tcdphpdrgm 1681tfnligesti rctsdphgng vwsspaprce lsvraghckt peqfpfaspt ipindfefpv 1741gtslnyecrp gyfgkmfsis clenlvwssv edncrrkscg pppepfngmv hintdtqfgs 1801tvnyscnegf rligspsttc lvsgnnvtwd kkapiceiis ceppptisng dfysnnrtsf 1861hngtvvtyqc htgpdgeqlf elvgersiyc tskddqvgvw ssppprcist nkctapeven 1921airvpgnrsf ftlteiirfr cqpgfvmvgs htvqcqtngr wgpklphcsr vcqpppeilh 1981gehtlshqdn fspgqevfys cepsydlrga aslhctpqgd wspeaprctv kscddflgql 2041phgrvllpln lqlgakvsfv cdegfrlkgr sashcvlagm kalwnssvpv ceqifcpnpp 2101ailngrhtgt pfgdipygke isyacdthpd rgmtfnlige ssirctsdpq gngvwsspap 2161rcelsvpaac phppkiqngh yigghvslyl pgmtisyicd pgyllvgkgf ifctdqgiws 2221qldhyckevn csfplfmngi skelemkkvy hygdyvtlkc edgytlegsp wsqcqaddrw 2281dpplakctsr thdalivgtl sgtiffilli iflswiilkh rkgnnahenp kevaihlhsq 2341ggssvhprtl qtneensrvl p (Seq. ID No. 3)

TABLE 4 Targets General Classes of Targets Microbes Polypeptides DNAAmino Acids Fungi Toxins RNA Prions Bacteria Lipids Parasites CytokinesVirus Cells Cellular debris Complement- associated moleculesComplement-Related Targets Immune complexes C3dg C4a C6 Factor B C3dkC4b C7 Factor D C3e C2 C8 Properdin Bb C4bp C9 C3 membrane attackMannose-Binding Lectin (MBL) complex C3a C1q MBL-Associated SerineProtease 1 (MASP1) C3b C1r MBL-Associated Serine Protease 2 (MASP2) iC3bC1s C5 C3c C4 C5a Infectious Disease-Related Targets LipopolysaccharidesCell invasion protein Intermedilysin Secreted effector protein sptP Zonaoccludens toxin Cholera enterotoxin Invasion protein sipASeeligeriolysin Actin polymerization protein Cysteine protease Iotatoxin component Serine protease RickA Ia Actin polymerization proteinCytolethal distending Ivanolysin Shiga toxin RickA toxin Adenosinemonophosphate- Cytolysin LepB Sphingomyelinase protein transferase vopSadenylate cyclase Cytotoxic necrotizing Lethal factor Staphylokinasefactor Adenylate cyclase ExoY Cytotoxin Leukotoxin StreptokinaseADP-ribosyltransferase Dermonecrotic toxin Listeriolysin Streptolysinenzymatic component Aerolysin Deubiquitinase Microbial collagenaseStreptopain Alpha-toxin Diphtheria toxin Outer membrane Suilysin proteinIcsA autotransporter Alveolysin Enterohemolysin Panton-ValentineSuperantigen Leucocidin F Alveolysin Enterotoxin Perfringolysin T3SSsecreted effector EspF Anthrolysin O Epidermal cell Pertussis toxinTetanus toxin differentiation inhibitor Arp2/3 complex-activatingExoenzyme Phospholipase Tir protein rickA Binary ADP-ribosyltransferaseExotoxin Plasminogen activator TolC CDT toxin Botulinum neurotoxinG-nucleotide Pneumolysin Toxic shock syndrome exchange factor toxin C2toxin, component II Guanine nucleotide Protective antigenZink-carboxypeptidase exchange factor sopE CagA Heat stable Proteinkinase Zink-carboxypeptidase enterotoxin Calmodulin-sensitive adenylateIgA-specific serine Pyolysin Zn-dependent cyclase endopeptidasepeptidase autotransporter Cell cycle inhibiting factor Inositolphosphate RTX toxin phosphatase sopB Other Molecular Targets G-CSF IL3IL10 MIP1a GM-CSF IL4 IL12 MIP1b M-CSF IL5 IFNa TGFb IL1a IL6 IFNb TNFaIL1b IL7 IFNg TNFb IL2 IL8 Self-antibodies Non-self antibodies PRP PRPcPRPsc PRPres Lipid & Cell Targets Circulating tumor cells very lowdensity lipid triglycerides Fatty acids (VLDL) Metastases high densitychylomicrons Cholesterol lipoprotein Eukaryotic cells low densityapolipoproteins lipoprotein

TABLE 5 Diseases and Conditions Cancers Acute Colorectal cancerMacroglobulinemia, Pleuropulmonary lymphoblastic Waldenström Blastoma,Childhood leukaemia (ALL) Acute myeloid Craniopharyngioma, ChildhoodMale Breast Cancer Pregnancy and Breast leukaemia (AML) CancerAdrenocortical Cutaneous T-Cell Lymphoma Malignant Fibrous PrimaryCentral Carcinoma Histiocytoma of Bone and Nervous System (CNS)Osteosarcoma Lymphoma AIDS-Related Kaposi Ductal Carcinoma In Situ(DCIS) Melanoma Prostate Cancer Sarcoma AIDS-Related Embryonal Tumors,Merkel Cell Carcinoma Rare cancers lymphoma Childhood Anal CancerEndometrial Cancer Mesothelioma Rectal Cancer Appendix CancerEpendymoma, Childhood Metastatic Squamous Renal cell carcinoma NeckCancer with Occult Primary Astrocytomas, Epithelial cancer Midline TractCarcinoma Renal Pelvis and Childhood Involving NUT Gene Ureter,Transitional Cell Cancer Atypical Esophageal Cancer Molar pregnancyRetinoblastoma Teratoid/Rhabdoid Tumor, Childhood Basal CellEsthesioneuroblastoma, Mouth and oropharyngeal RhabdomyosarcomaCarcinoma Childhood cancer Bile duct cancer Ewing sarcoma MultipleEndocrine Salivary Gland Cancer Neoplasia Syndromes, Childhood Bladdercancer Extragonadal Germ Cell Tumor Multiple Sarcoma Myeloma/Plasma CellNeoplasm Bone cancer Extrahepatic Bile Duct Cancer Mycosis FungoidesSecondary cancers Bowel cancer Eye Cancer Myelodysplastic SézarySyndrome Syndromes Brain Stem Glioma, Gallbladder CancerMyelodysplastic/ Skin Cancer Childhood Myeloproliferative NeoplasmsBrain tumours Gastric cancer Myeloproliferative Skin cancer (nonDisorders, Chronic melanoma) Breast cancer Gastrointestinal CarcinoidTumor Nasal Cavity and Small Cell Lung Cancer Paranasal Sinus CancerBronchial Tumors, Germ Cell Tumor Nasopharyngeal cancer Small IntestineCancer Childhood Burkitt Lymphoma Gestational trophoblasticNeuroblastoma Soft Tissue Sarcoma tumours (GTT) Cancer of unknown GliomaNon-Hodgkin Lymphoma Squamous Cell primary Carcinoma Cancer spread toHairy cell leukaemia Non-Small Cell Lung Squamous Neck bone CancerCancer with Occult Primary, Metastatic Cancer spread to Head and neckcancer Oesophageal cancer Stomach (Gastric) brain Cancer Cancer spreadto Heart Cancer, Childhood Oral Cancer Stomach cancer liver Cancerspread to Hepatocellular (Liver) Cancer Oral Cavity Cancer T-CellLymphoma, lung Cutaneous - see Mycosis Fungoides and Sézary SyndromeCarcinoid Tumor Histiocytosis, Langerhans Cell Oropharyngeal CancerTesticular cancer Carcinoma of Hodgkin Lymphoma Osteosarcoma (BoneThroat Cancer Unknown Primary Cancer) Cardiac (Heart) HypopharyngealCancer Osteosarcoma and Thymoma and Thymic Tumors, Childhood MalignantFibrous Carcinoma Histiocytoma Central Nervous Intraocular MelanomaOvarian Cancer Thyroid Cancer System Atypical Teratoid/Rhabdoid Tumor,Childhood Central Nervous Islet Cell Tumors, Pancreatic PancreaticCancer Transitional Cell System Embryonal Neuroendocrine Tumors Cancerof the Renal Tumors, Childhood Pelvis and Ureter Central Nervous Kidneycancer Pancreatic Unknown primary System, Childhood NeuroendocrineTumors cancer (Islet Cell Tumors) Cervical cancer Langerhans CellHistiocytosis Papillomatosis, Childhood Ureter and Renal Pelvis,Transitional Cell Cancer Chordoma, Laryngeal Cancer ParagangliomaUrethral Cancer Childhood Choriocarcinoma Leukemia Parathyroid CancerUterine Cancer, Endometrial Chronic Lip and Oral Cavity Cancer PenileCancer Uterine Sarcoma Lymphocytic Leukemia (CLL) Chronic myeloid Livercancer Pharyngeal Cancer Vaginal cancer leukaemia (CML) Chronic LobularCarcinoma In Situ (LCIS) Pheochromocytoma Vulvar CancerMyeloproliferative Disorders Colon cancer Low Malignant Potential TumorPituitary Tumor Waldenström Macroglobulinemia Lymphoma Lung CancerPlasma Cell Wilms Tumor Neoplasm/Multiple Myeloma Complement and ImmuneComplex-Related Diseases Age-related ANCA-associated vasculitisGlomerulonephritis - MYH9-related disease macular (IncludesPauci-immune) sparse hair - telangiectasis degeneration Atypicalhemolytic Anti-glomerular basement Goodpasture's sndrome Nail-patellasyndrome uremic syndrome membrane disease (Goodpasture's) AutoimmuneArthus Reaction Granulomatosis with Nail-patella-like renal hemolyticanemia polyangiitis (ANCA and disease Wegeners) C1 inhibitor AsthmaGuillain-Barre syndrome Nephritis deficiency C1q deficiency Atypicalhemolytic uremic Hemolytic angioedema Non-amyloid syndrome (HAE)monoclonal immunoglobulin deposition disease C1r deficiency Autoimmuneinner ear disease Henoch-Schonlein Pauci-immune (AIED) Sensorineuralhearing loss purpura glomerulonephritis C1s deficiency Autoimmuneuveitis HIVICK Pediatric systemic lupus erythematosus C2 deficiencyAutosomal dominant Hypersensitivty vasculitis Pierson syndromeintermediate Charcot-Marie- Tooth disease type E C3 deficiency Behçetdisease Hypocomplementemic Polyarteritis urticarial vasculitis C4deficiency Berger (IgA) Nephropathy Idiopathic membranous polyarteritisnodosa glomerulonephritis C5 deficiency Buergers disease Idiopathicnephrotic Polymyalgia syndrome rheumatica C6 deficiency Central nervoussystem vasculitis IgA nephropathy (Berger's Polymyositis disease) C7deficiency Choroiditis IgA Polymyositis/ nephropathy/vasculitisdermatomyositis (Henoch-Schonlein purpura) C8 deficiency Chronicdemyelinating Immune Poststaphilococcal polyneuropathy (CIDP)thrombocytopenia glomerulonephritis C9 deficiency Churg-strauss syndromeImmunobullous diseases Poststeptococcal glomerulonephritis CD55deficiency Cogan's syndrome Immunotactoid or Primary fibrillaryglomerulopathy membranoproliferative glomerulonephritis CD59 deficiencyCollagen type III glomerulopathy Infection-related Rapidly progressiveglomerulonephritis glomerulonephritis (Crescentic) Complement FactorCongenital and infantile Inflammatory myopathies Rapidly progressive Ideficiency nephrotic syndrome glomerulonephritis (RPGN) Complementfactor- Congenital membranous Juvenile dermatomyositis Rasmussensyndrome H related 1(CFHR1) nephropathy due to maternal deficiencyanti-neutral endopeptidase alloimmunization Complement factor-Cryoglobulinaemia/Cold Juvenile polymyositis Reactive arthritis Hrelated 3(CFHR3) agglutinin diease deficiency CR3/CR4 Cryoglobulinemicvasculitis Kawasaki disease Relapsing defieciency polychondritis(leukocyte adhesion deficiency 1) Factor B deficiency Cutaneousvasculitis Lipoprotein Renal amyloidosis glomerulopathy Factor Ddeficiency Demyelinating myopathies Lupus nephritis Reynolds syndrome(paraprotein associated) Factor H deficiency Denys-Drash syndrome Lupusnephropathy Rheumatoid arthritis Factor I deficiency Dermatomyositis MayHegglin anomaly Sarcoidosis (Nesnier Boeck Schuamann Disease) Ficolin 3deficiency Dermatomyositis Membranoglomerular Schimke immuno- nephritisosseous dysplasia MASP2 deficiency Diabetic nephropathyMembranoproliferative Scleroderma glomerulonephritis MBL deficiencyDrug-induced immune complex Membranoproliferative Sebastian syndromevasculitis glomerulonephritis Type I (MPGN Type I) Non-alcoholicEosinophilic granulomatosis with Membranoproliferative Secondarysteatohepatitis polyangiitis (Churgg-Strauss) glomerulonephritis Type IIamyloidosis (Dense Deposit Disease, MPGN Type II) Paroxysmal EpsteinSyndrome Membranoproliferative Severe or recurring nocturnalglomerulonephritis Type C diff colitis hemoglobinuria III (MPGN TypeIII) Properdin deficiency Essential mixed cryoglobulinemia MembranouseSjogren's syndrome glomerulonephritis Action myoclonus - FamilialMediterranean fever Menieres disease Staphylococcal or renal failurestreptococcal sepsis syndrome Acute respiratory Familial renalamyloidosis Microscopic polyangiitis Stiff person syndrome diseasesyndrome (ARDS)/Severe acute respiratory syndrome (SARS) Acute serumFamilial steroid-resistant Minimal change disease Systemic lupussickness nephrotic syndrome with erythematosus sensorineural deafnessAdult-onset Still Farmer's lung Mixed connective tissue Systemicsclerosis disease disease Age-related Fechtner Syndrome Mostly largevessel Takayasu arteritis macular vasculitis degeneration AL amyloidosisFibronectin glomerulopathy mostly medium vessel Toxic epidermalvasculitis necrolysis (Stevens Johnson syndrome) Alport's syndromeFibrosing alveolitis Mostly small vessel Transplantation/ vsculitisreperfusion (solid organ) Alzheimer's disease Focal segmental glomerularMuckle-Wells syndrome Vasculitis Amyloidosis (AL, Focal segmentalMyasthenia gravis Wegener's AA, MIDD, Other) glomerulosclerosisgranulomatosis Giant cell arteritis Frasier syndrome Galloway-Mowatsyndrome Type 1 diabetes Myasthenia gravis Graves' disease Perniciousanemia Crohn's disease alopecia areata thrombocytopenic Primary biliarypurpura cirrhosis Ulcerative colitis autoimmune hepatitis Guillain-Barresyndrome Psoriasis Inflammatory autoimmune deramtomyositis Autoimmunemyocarditis Rheumatoid arthritis bowel syndrome Multiple sclerosisJuvenile idiopathic arthritis Autoimmune pemphigus Vitiligo EnzymeDeficiencies & Vascular Diseases 2,4-dienoyl-CoA Fabry disease (1:80,000to Isobutyryl-CoA Peripheral reductase 1:117,000) dehydrogenaseneuropathy deficiency 2-Methyl-3-hydroxy Familial hypercholesterolemiaIsovaleric acidemia Peroxisomal disorders butyric aciduria (1:500)(1:50,000; e.g., Zellweger syndrome, neonatal adrenoleukodystrophy,Refsum's disease) 2-methylbutyryl- Familial myocardial infarct/strokeLactase deficiency Phenylketonuria CoA dehydrogenase (common)3-hydroxy-3- Fatty acid oxidation disorders Lesch-Nyhan syndrome Primaryhyperoxaluria methylglutaryl (1:10,000) (HMG) aciduria3-methylglutaconic Galactokinase deficiency Lipoprotein lipase Propionicacidemia aciduria deficiency (rare) 3-oxothiolase Galactose epimeraselong-chain l-3-hdroxyacyl- Recurrent emesis deficiency CoA dehydrogenase(1:100,000) 4-hydroxybutyric Galactosemia Lysinuric protein Short-chainacyl-CoA aciduria intolerance (rare) dehydrogenase 5,10-methylenetetra-Galactosemia (1:40,000) Lysinuric protein Sucrase-isomaltase hydrofolatereductase intolerance (rare) deficiency (rare) deficiency (common)5-Oxoprolinuria Gaucher's disease Malonic acidemia Symptoms of(pyroglutamic pancreatitis aciduria) Abetalipoproteinemia Glutaricacidemia type I Maple syrup urine disease Transferase deficient (rare)galactosemia (Galactosemia type 1) Acute Intermittent Glutaric acidemiaType II Medium chain acyl-CoA Trifunctional protein Porphyriadehydrogenase deficiency Alkaptonuria Glutathione SynthetaseMedium/short chain L-3- Tyrosinemia type 1 Deficiency w/5-oxoprolinuriahydroxy acyl-CoA dehydrogenase Argininemia Glutathione SynthetaseMedum-chain ketoacyl- Tyrosinemia type 2 Deficiency w/o 5-oxoprolinuriacoA thiolase argininosuccinate Glycogenolysis disorders MetachromaticTyrosinemia type 3 aciduria (1:20,000) leukodystrophy (1:100,000) BenignGlycogenosis, type I (1:70,000) Metachromatic Upward gaze paralysishyperphenylalaninemia leukodystrophy (1:100,000) beta ketothiolaseHemolytic anemia due to Methylmalonic acidemia Very long chain acyl-deficiency adenylate kinase deficiency (Cbl C) CoA dehydrogenaseBiopterin cofactor Hemolytic anemia due to Methylmalonic acidemia WilsonDisease biosynthesis defects deficiency in Glucose 6 (Cbl D) phosphatedehydrogenase Biopterin cofactor Hemolytic anemia due to Methylmalonicacidemia Aicardi-Goutieres regeneration diphosphoglycerate mutase(vitamin b12 non- Syndrome (may be an defects deficiency responsive)allelic form of CLE) biotin-unresponsive Hemolytic anemia due toMethylmalonic acidemia Cutaneous lupus 3-methylcrotonyl- erythrocyteadenosine w/0 homocystinuria erythematosus CoA carboxylase deaminaseoverproduction deficiency Carbamoyl Hemolytic anemia due toMethylmalonic aciduria Dermatitis phosphate glucophosphate isomerase andhomocystinuria herpetiformis synthetase deficiency Carnitine Hemolyticanemia due to Mitochondrial disorders hemophilia A acylcarnitineglutathione reductase deficiency (1:30,000) translocase CarnitineHemolytic anemia due to Mitochondrial disorders hemophilia Bpalmitoyltransferase I glyceraldehyde-3-phosphate (1:30,000; e.g.,dehydrogenase deficiency cytochrome-c oxidase deficiency; MELASsyndrome; Pearson's syndrome [all rare]) Carnitine Hemolytic anemia dueto Mitochondrial disorders Idiopathic steroid palmitoyltransferase IIpyrimidine 5′ nucleotidase (1:30,000; e.g., Leigh sensitive nephroticdeficiency disease, Kearns-Sayre syndrome (same as syndrome [rare])focal segmental glomerulaosclerosis) Carnitine uptake Hemolytic anemiadue to red cell Mitochondrial disorders Immune defect pyruvate kinasedeficiency (1:30,000; e.g., lipoamide thrombocytopenic dehydrogenasedeficiency purpura [rare]) citrullinemia type I HHH syndrome (rare)Mitochondrial disorders Myasthenia gravis (1:30,000; e.g., Pearson'ssyndrome [rare]) Citrullinemia type II homocysteinuria Multiplecarboxylase Oligoarticular juvenile (holocarboxylase arthritissynthetase) Congenital Homocystinuria (1:200,000) Multiple carboxylaseScleroderma disorders of deficiency (e.g., glycosylation (rare)holocarboxylase synthetase [rare]) and biotinidase deficiencies(1:60,000) D-2-hydroxyglutaric hyperammonemia/ornithinemia/ Musclecramps/spasticity Solar urticaria (maybe aciduria citrullinemia(ornithine protophyria transporter defect) erythema)D-2-hydroxyglutaricaciduria Hyperlipoproteinemia, types I Myoadenylatedeaminase Thrombotic (rare) and IV (rare) deficiency (1:100,000)thrombocytopenic purpura Enteropeptidase Hypermethioninemia due toNiemann-Pick disease, Tubulointerstitial deficiency (rare) glycineN-methyltransferase type C (rare) nephritis with Uveitis/ deficiencyATIN Ethylmalonic Hypermethioninemia Nonketotic Von willebrandencephalopathy encephalopathy due to hyperglycinemia disease adenosinekinase deficiency Hyperprolinemia Infectious Diseases & agentsAcinetobacter Dengue haemorrhagic fever Infection-induced Sepsis immunecomplex vasculitis Arcobacter butzleri Disseminated infection withKlebsiella Serratia infection - blood mycobacterium avium complex -infection blood infection Arcobacter E. coli Leprosy/Hansen's diseaseStaphylococcus cryaerophilus Aureus infection - blood infectionArcobacter Enterobacter Malaria Stenotrophomonas infection - bloodmaltophilia - blood infection infection Bacteremia EnterococcusMeningococcus Streptococcal Group A invasive disease - blood infectionBacterial Glanders - blood infection Methicillin Resistant Streptococcusendocarditis Staphylococcus Aureus pneumoniae Campylobacter GonorrheaPseudomonas Streptococcus fetus infection - pyogenes blood infectionCampylobacter Hepatitis Rhodococcus equi - blood Trypanosomiasis jejuniinfection - infection blood infection Candida Human ImmunodeficiencyVirus Salmonella Yellow fever Coagulase-negative Staphylococcus

TABLE 6 Exogenous antigens General Classes of Exogenous antigens Ankyrinrepeat proteins Fibronectins Lyases Antibodies Complement receptorsGPI-linked polypeptides Nanobodies Aptamers Cyclic peptides HEAT repeatproteins Nucleic Acids ARM repeat proteins DARPins HydrolasesPolypeptides Carbohydrates DNAses Kinases Single-chain variablefragments (scFv) Cell surface receptors Enzymes LipoproteinsTetratricopeptide repeat proteins Complement-Related Exogenous antigensC1 inhibitor C4 binding protein CR3 Factor I C3 Beta chain Receptor CD59CR4 Homologous restriction factor C3aR CR1 Decay-accelerating factorMembrane cofactor protein (DAF) (MCP) C3eR CR2 Factor H PRELP Enzymestriacylglycerol lipase bile-acid-CoA hydrolase feruloyl esterasephosphatidate phosphatase (S)-methylmalonyl-CoAbis(2-ethylhexyl)phthalate formyl-CoA hydrolasephosphatidylglycerophosphatase hydrolase esterase [acyl-carrier-protein]bisphosphoglycerate fructose-bisphosphatase phosphatidylinositoldeacylase phosphodiesterase phosphatase [phosphorylase] Carboxylic-EsterHydrolases fumarylacetoacetase phosphodiesterase I phosphatase1,4-lactonase carboxymethylenebutenolidase fusarinine-C phosphoglyceratephosphatase ornithinesterase 11-cis-retinyl-palmitatecellulose-polysulfatase galactolipase phosphoglycolate phosphatasehydrolase 1-alkyl-2- cephalosporin-C deacetylase gluconolactonasephosphoinositide phospholipase acetylglycerophosphocholine C esterase2′-hydroxybiphenyl-2- cerebroside-sulfatase glucose-1-phosphatasephospholipase A1 sulfinate desulfinase 2-pyrone-4,6-dicarboxylatecetraxate benzylesterase glucose-6-phosphatase phospholipase A2lactonase 3′,5′-bisphosphate chlorogenate hydrolase glutathionethiolesterase phospholipase C nucleotidase 3-hydroxyisobutyryl-CoAchlorophyllase glycerol-1-phosphatase phospholipase D hydrolase3′-nucleotidase cholinesterase glycerol-2-phosphatasephosphonoacetaldehyde hydrolase 3-oxoadipate enol-lactonasecholine-sulfatase glycerophosphocholine phosphonoacetate hydrolasephosphodiesterase 3-phytase choloyl-CoA hydrolase Glycosidases, i.e.enzymes phosphonopyruvate hydrolase that hydrolyse O- and S- glycosylcompounds 4-hydroxybenzoyl-CoA chondro-4-sulfatase glycosulfatasephosphoprotein phosphatase thioesterase 4-methyloxaloacetatechondro-6-sulfatase Glycosylases Phosphoric-diester hydrolases esterase4-phytase citrate-lyase deacetylase histidinol-phosphatasePhosphoric-monoester hydrolases 4-pyridoxolactonase cocaine esterasehormone-sensitive lipase Phosphoric-triester hydrolases 5′-nucleotidasecutinase Hydrolysing N-glycosyl phosphoserine phosphatase compounds6-acetylglucose deacetylase cyclamate sulfohydrolase HydrolysingS-glycosyl poly(3-hydroxybutyrate) compounds depolymerase6-phosphogluconolactonase Cysteine endopeptidases hydroxyacylglutathionepoly(3-hydroxyoctanoate) hydrolase depolymerase a-amino-acid esteraseCysteine-type hydroxybutyrate-dimer polyneuridine-aldehyde esterasecarboxypeptidases hydrolase a-Amino-acyl-peptide D-arabinonolactonasehydroxymethylglutaryl-CoA protein-glutamate hydrolases hydrolasemethylesterase acetoacetyl-CoA hydrolase deoxylimonate A-ring-iduronate-2-sulfatase quorum-quenching N-acyl- lactonase homoserinelactonase acetoxybutynylbithiophene dGTPase inositol-phosphateretinyl-palmitate esterase deacetylase phosphatase acetylajmalineesterase dihydrocoumarin hydrolase juvenile-hormone esterase Serinedehyrdatase or serine hydroxymethyl transferase acetylalkylglycerolDipeptidases kynureninase Serine endopeptidases acetylhydrolaseacetylcholinesterase Dipeptide hydrolases L-arabinonolactonaseserine-ethanolaminephosphate phosphodiesterase acetyl-CoA hydrolaseDipeptidyl-peptidases and limonin-D-ring-lactonase Serine-typecarboxypeptidases tripeptidyl-peptidases acetylesteraseDiphosphoric-monoester lipoprotein lipase S-formylglutathione hydrolasehydrolases acetylpyruvate hydrolase disulfoglucosamine-6-sulfataseL-rhamnono-1,4-lactonase sialate O-acetylesterase acetylsalicylatedeacetylase dodecanoyl-[acyl-carrier- lysophospholipase sinapineesterase protein] hydrolase acetylxylan esterase Endodeoxyribonucleasesmannitol-1-phosphatase Site specific producing 3′-endodeoxyribonucleases: phosphomonoesters cleavage is not sequencespecific acid phosphatase EndodeoxyribonucleasesMetallocarboxypeptidases Site-specific producing 5′-endodeoxyribonucleases that phosphomonoesters are specific for alteredbases. Acting on acid anhydrides to Endopeptidases of unknownMetalloendopeptidases. Site-specific catalyse transmembrane catalyticmechanism endodeoxyribonucleases: movement of substances cleavage issequence specific Acting on acid anhydrides to Endoribonucleasesproducing methylphosphothioglycerate sphingomyelin facilitate cellularand 3′-phosphomonoesters phosphatase phosphodiesterase subcellularmovement Acting on GTP to facilitate Endoribonucleases producingmethylumbelliferyl-acetate S-succinylglutathione hydrolase cellular andsubcellular 5′-phosphomonoesters deacetylase movement Acting onphosphorus- Endoribonucleases that are monoterpene e-lactonesteroid-lactonase nitrogen bonds active with either ribo- or hydrolasedeoxyribonucleic acids and produce 3′- phosphomonoesters Acting onsulfur-nitrogen Endoribonucleases that are N-acetylgalactosamine-4-sterol esterase bonds active with either ribo- or sulfatasedeoxyribonucleic acids and produce 5′- phosphomonoesters actinomycinlactonase Enzymes acting on acid N-acetylgalactosamine-6-steryl-sulfatase anhydrides sulfatase acylcarnitine hydrolase EnzymesActing on carbon- N- succinyl-CoA hydrolase carbon bondsacetylgalactosaminoglycan deacetylase acyl-CoA hydrolase Enzymes actingon carbon- N-acetylglucosamine-6- sucrose-phosphate phosphatase nitrogenbonds, other than sulfatase peptide bonds acylglycerol lipase Enzymesacting on carbon- N-sulfoglucosamine sugar-phosphatase phosphorus bondssulfohydrolase acyloxyacyl hydrolase Enzymes acting on carbon-oleoyl-[acyl-carrier-protein] Sulfuric-ester hydrolases sulfur bondshydrolase acylpyruvate hydrolase Enzymes Acting on ether bonds Omegapeptidases tannase ADAMTS13 Enzymes acting on halide orsellinate-depsideThioester hydrolases bonds hydrolase Adenosine deaminase Enzymes actingon peptide oxaloacetase Thioether and trialkylsulfonium bonds(peptidases) hydrolases adenylyl-[glutamate- Enzymes acting onpalmitoyl[protein] hydrolase Threonine endopeptidases ammonia ligase]phosphorus-nitrogen bonds hydrolase ADP-dependent medium- Enzymes actingon sulfur- palmitoyl-CoA hydrolase thymidine phosphorylasechain-acyl-CoA hydrolase nitrogen bonds ADP-dependent short-chain-Enzymes acting on sulfur-sulfur pectinesterase trehalose-phosphataseacyl-CoA hydrolase bonds ADP-phosphoglycerate Ether hydrolases. Peptidylpeptide hydrolases triacetate-lactonase phosphatase alkaline phosphataseExodeoxyribonucleases Peptidyl-amino-acid Triphosphoric-monoesterproducing 5′- hydrolases hydrolases phosphomonoestersall-trans-retinyl-palmitate Exonucleases that are activePeptidylamino-acid trithionate hydrolase hydrolase with either ribo- orhydrolases or acylamino- deoxyribonucleic acids and acid hydrolasesproduce 3′- phosphomonoesters aminoacyl-tRNA hydrolase Exonucleases thatare active Peptidyl-dipeptidases tropinesterase with either ribo- ordeoxyribonucleic acids and produce 5′- phosphomonoesters AminopeptidasesExoribonucleases producing 3′- phenylacetyl-CoA hydrolase ubiquitinthiolesterase phosphomonoesters arylesterase Exoribonucleases producing5′- Phenylalanine ammonia UDP-sulfoquinovose synthase phosphomonoesters.lyase arylsulfatase Factor IX Phenylalanine hydroxylase uricaseAsparaginase Factor VIII pheophorbidase uronolactonase Asparticendopeptidases fatty-acyl-ethyl-ester synthase phloretin hydrolasewax-ester hydrolase b-diketone hydrolase phorbol-diester hydrolasexylono-1,4-lactonase

TABLE 7 Selected Diseases, Exogenous antigens and Targets CategoryDisease Exogenous antigen Target Amyloidoses AA Amyloidosis an anantibody-like binder to serum Serum amyloid A protein and amyloid Aprotein or serum amyloid P amyloid placques component Amyloidoses beta2microglobulin amyloidosis an an antibody-like binder to beta-2 Beta2microglobulin or amyloid microglobulin or serum amyloid P placquescomponent Amyloidoses Light chain amyloidosis an an antibody-like binderto light chain, Antibody light chain or amyloid serum amyloid Pcomponent placques Cell clearance Cancer an an antibody-like binder toCD44 a circulating tumor cell Cell clearance Cancer an an antibody-likebinder to EpCam a circulating tumor cell Cell clearance Cancer an anantibody-like binder to Her2 a circulating tumor cell Cell clearanceCancer an an antibody-like binder to EGFR a circulating tumor cell Cellclearance Cancer (B cell) an an antibody-like binder to CD20 a cancerousB cell Cell clearance Cancer (B cell) an an antibody-like binder to CD19a cancerous B cell Clearance Ab Antiphospholipid syndromebeta2-glycoprotein-1 pathogenic self-antibody againstbeta2-glycoprotein-1 Clearance Ab Catastrophic antiphospholipidbeta2-glycoprotein-1 pathogenic self-antibody syndrome againstbeta2-glycoprotein-1 Clearance Ab Cold agglutinin disease I/i antigenPathogenic self-antibody against I/i antigen Clearance Ab Goodpasturesyndrome a3 NC1 domain of collagen (IV) pathogenic self-antibody againsta3 NC1 domain of Collagen (IV) Clearance Ab Immune thrombocytopeniaPlatelet Glycoproteins (Ib-IX, IIb-IIIa, IV, Ia- pathogenicself-antibody purpura IIa) against platelet glycoprotein Clearance AbMembranous Nephropathy Phospholipase A2 receptor pathogenicself-antibody against phospholipase A2 receptor Clearance Ab Warmantibody hemolytic anemia Glycophorin A, glycophorin B, and/orpathogenic self-antibody glycophorin C, Rh antigen against glycophorinsand/or Rh antigen Complement Age-related macular degeneration a suitablecomplement regulatory protein active complement Complement Atypicalhemolytic uremic complement factor H, or a suitable active complementsyndrome complement regulatory protein Complement Autoimmune hemolyticanemia a suitable complement regulatory molecule active complementComplement Complement Factor I deficiency Complement factor I, asuitable active complement complement regulatory protein ComplementNon-alcoholic steatohepatitis a suitable complement regulatory moleculeactive complement Complement Paroxysmal nocturnal a suitable complementregulatory protein active complement hemoglobinuria Enzyme3-methylcrotonyl-CoA carboxylase 3-methylcrotonyl-CoA carboxylase3-hydroxyvalerylcarnitine, 3- deficiency methylcrotonylglycine (3-MCG)and 3-hydroxyisovaleric acid (3- HIVA) Enzyme Acute IntermittentPorphyria Porphobilinogen deaminase Porphobilinogen Enzyme Acutelymphoblastic leukemia Asparaginase Asparagine Enzyme Acute lymphocyticleukemia, Asparaginase Asparagine acute myeloid leukemia Enzyme Acutemyeloblastic leukemia Asparaginase Asparagine Enzyme Adenine adeninephosphoribosyltransferase Insoluble purine 2,8-phosphoribosyltransferase dihydroxyadenine deficiency Enzyme Adenosinedeaminase deficiency Adenosine deaminase Adenosine EnzymeAfibrinogenomia FI enzyme replacement Enzyme Alcohol poisoning Alcoholdehydrogenase/oxidase Ethanol Enzyme Alexander's disease FVII enzymereplacement Enzyme Alkaptonuria homogentisate oxidase homogentisateEnzyme Argininemia Ammonia monooxygenase ammonia Enzymeargininosuccinate aciduria Ammonia monooxygenase ammonia Enzymecitrullinemia type I Ammonia monooxygenase ammonia Enzyme Citrullinemiatype II Ammonia monooxygenase ammonia Enzyme Complete LCAT deficiency,Fish- Lecithin-cholesterol acyltransferase (LCAT) Cholesterol eyedisease, atherosclerosis, hypercholesterolemia Enzyme Cyanide poisoningThiosulfate-cyanide sulfurtransferase Cyanide Enzyme DiabetesHexokinase, glucokinase Glucose Enzyme Factor II Deficiency FII enzymereplacement Enzyme Familial hyperarginemia Arginase Arginine EnzymeFibrin Stabilizing factor Def. FXIII enzyme replacement Enzyme Glutaricacidemia type I lysine oxidase 3-hydroxyglutaric and glutaric acid(C5-DC), lysine Enzyme Gout Uricase Uric Acid Enzyme Gout -hyperuricemia Uricase Uric acid (Urate crystals) Enzyme Hageman Def.FXII enzyme replacement Enzyme Hemolytic anemia due to pyrimidine 5′nucleotidase pyrimidines pyrimidine 5′ nucleotidase deficiency EnzymeHemophilia A Factor VIII Thrombin (factor II a) or Factor X EnzymeHemophilia B Factor IX Factor XIa or Factor X Enzyme Hemophilia C FXIenzyme replacement Enzyme Hepatocellular carcinoma, Arginine deiminaseArginine melanoma Enzyme Homocystinuria Cystathionine B synthasehomocysteine Enzyme hyperammonemia/ornithinemia/ Ammonia monooxygenaseAmmonia citrullinemia (ornithine transporter defect) Enzyme Isovalericacidemia Leucine metabolizing enzyme leucine Enzyme Lead poisoningd-aminolevulinate dehydrogenase lead Enzyme Lesch-Nyhan syndrome UricaseUric acid Enzyme Maple syrup urine disease Leucine metabolizing enzymeLeucine Enzyme Methylmalonic acidemia (vitamin methylmalonyl-CoA mutasemethylmalonate b12 non-responsive) Enzyme Mitochondrial thymidinephosphorylase thymidine neurogastrointestinal encephalomyopathy EnzymeMitochondrial Thymidine phosphorylase Thymidine neurogastrointestinalencephalomyopathy (MNGIE) Enzyme Owren's disease FV enzyme replacementEnzyme p53-null solid tumor Serine dehyrdatase or serine serinehydroxymethyl transferase Enzyme Pancreatic adenocarcinoma Asparaginaseasparagine Enzyme Phenylketonuria Phenylalanine hydroxylase,phenylalanine Phenylalanine ammonia lyase Enzyme Primary hyperoxaluriaOxalate oxidase Oxalate Enzyme Propionic acidemia Propionate conversionenzyme? Proprionyl coA Enzyme Purine nucleoside phosphorylase Purinenucleoside phosphorylase Inosine, dGTP deficiency Enzyme Stuart-PowerDef. FX enzyme replacement Enzyme Thrombotic Thrombocytopenic ADAMTS13ultra-large von willebrand Purpura factor (ULVWF) Enzyme Transferasedeficient galactose dehydrogenase Galactose-1-phosphate galactosemia(Galactosemia type 1) Enzyme Tyrosinemia type 1 tyrosine phenol-lyasetyrosine Enzyme von Willebrand disease vWF enzyme replacement ICclearance IgA Nephropathy Complement receptor 1 Immune complexes ICclearance Lupus nephritis Complement receptor 1 immune complex ICclearance Systemic lupus erythematosus Complement receptor 1 immunecomplex Infectious Anthrax (B. anthracis) infection an an antibody-likebinder to B. anthracis B. anthracis surface protein Infectious C.botulinum infection an an antibody-like binder to C. botulinum C.botulinum surface protein Infectious C. difficile infection anantibody-like binder to C. difficile C. difficile surface proteinInfectious Candida infection an antibody-like binder to candida surfacecandida protein Infectious E. coli infection an antibody-like binder toE. coli surface E. coli protein Infectious Ebola infection anantibody-like binder to Ebola surface Ebola protein Infectious HepatitisB (HBV) infection an antibody-like binder to HBV surface HBV proteinInfectious Hepatitis C (HCV) infection an antibody-like binder to HCVsurface HCV protein Infectious Human immunodeficiency virus anantibody-like binder to HIV envelope HIV (HIV) infection proteins or CD4or CCR5 or Infectious M. tuberculosis infection an antibody-like binderto M. tuberculosis M. tuberculosis surface protein Infectious Malaria(P. falciparum) infection an antibody-like binder to P. falciparum P.falciparum surface protein Lipid Hepatic lipase deficiency, Hepaticlipase (LIPC) Lipoprotein, intermediate hypercholesterolemia density(IDL) Lipid Hyperalphalipoproteinemia 1 Cholesteryl ester transferprotein(CETP) Lipoprotein, high density (HDL) Lipid hypercholesterolemiaan antibody-like binder to low-density LDL lipoprotein (LDL), LDLreceptor Lipid hypercholesterolemia an antibody-like binder tohigh-density HDL lipoprotein (HDL) or HDL receptor Lipid lipoproteinlipase deficiency lipoprotein lipase chilomicrons and very low densitylipoproteins (VLDL) Lipid Lipoprotein lipase deficiency, lipoproteinlipase (LPL) Lipoprotein, very low density disorders of lipoprotein(VLDL) metabolism Lysosomal storage Aspartylglucosaminuria (208400)N-Aspartylglucosaminidase glycoproteins Lysosomal storageCerebrotendinous xanthomatosis Sterol 27-hydroxylase lipids,cholesterol, and bile acid (cholestanol lipidosis; 213700) Lysosomalstorage Ceroid lipofuscinosis Adult form Palmitoyl-proteinthioesterase-1 lipopigments (CLN4, Kufs' disease; 204300) Lysosomalstorage Ceroid lipofuscinosis Infantile Palmitoyl-protein thioesterase-1lipopigments form (CLN1, Santavuori-Haltia disease; 256730) Lysosomalstorage Ceroid lipofuscinosis Juvenile form Lysosomal transmembrane CLN3protein lipopigments (CLN3, Batten disease, Vogt- Spielmeyer disease;204200) Lysosomal storage Ceroid lipofuscinosis Late infantile Lysosomalpepstatin-insensitive peptidase lipopigments form (CLN2,Jansky-Bielschowsky disease; 204500) Lysosomal storage Ceroidlipofuscinosis Progressive Transmembrane CLN8 protein lipopigmentsepilepsy with intellectual disability (600143) Lysosomal storage Ceroidlipofuscinosis Variant late Transmembrane CLN6 protein lipopigmentsinfantile form (CLN6; 601780) Lysosomal storage Ceroid lipofuscinosisVariant late Lysosomal transmembrane CLN5 protein lipopigments infantileform, Finnish type (CLN5; 256731) Lysosomal storage Cholesteryl esterstorage disease lisosomal acid lipase lipids and cholesterol (CESD)Lysosomal storage Congenital disorders of N- Phosphomannomutase-2N-glycosylated protein glycosylation CDG Ia (solely neurologic andneurologic- multivisceral forms; 212065) Lysosomal storage Congenitaldisorders of N- Mannose (Man) phosphate (P) isomerase N-glycosylatedprotein glycosylation CDG Ib (602579) Lysosomal storage Congenitaldisorders of N- Dolicho-P-Glc: Man9GlcNAc2-PP-dolichol N-glycosylatedprotein glycosylation CDG Ic (603147) glucosyltransferase Lysosomalstorage Congenital disorders of N- Dolicho-P-Man:Man5GlcNAc2-PP-dolichol N-glycosylated protein glycosylation CDG Id(601110) mannosyltransferase Lysosomal storage Congenital disorders ofN- Dolichol-P-mannose synthase N-glycosylated protein glycosylation CDGIe (608799) Lysosomal storage Congenital disorders of N- Proteininvolved in mannose-P-dolichol N-glycosylated protein glycosylation CDGIf (609180) utilization Lysosomal storage Congenital disorders of N-Dolichyl-P-mannose: Man-7-GlcNAc-2-PP- N-glycosylated proteinglycosylation CDG Ig (607143) dolichyl-α-6-mannosyltransferase Lysosomalstorage Congenital disorders of N- Dolichyl-P-glucose:Glc-1-Man-9-GlcNAc-2- N-glycosylated protein glycosylation CDG Ih(608104) PP-dolichyl-α-3-glucosyltransferase Lysosomal storageCongenital disorders of N- α-1,3-Mannosyltransferase N-glycosylatedprotein glycosylation CDG Ii (607906) Lysosomal storage Congenitaldisorders of N- Mannosyl-α-1,6-glycoprotein-β-1,2-N- N-glycosylatedprotein glycosylation CDG IIa (212066) acetylglucosminyltransferaseLysosomal storage Congenital disorders of N- Glucosidase IN-glycosylated protein glycosylation CDG IIb (606056) Lysosomal storageCongenital disorders of N- GDP-fucose transporter-1 N-glycosylatedprotein glycosylation CDG IIc (Rambam- Hasharon syndrome; 266265Lysosomal storage Congenital disorders of N- β-1,4-GalactosyltransferaseN-glycosylated protein glycosylation CDG IId (607091) Lysosomal storageCongenital disorders of N- Oligomeric Golgi complex-7 N-glycosylatedprotein glycosylation CDG IIe (608779) Lysosomal storage Congenitaldisorders of N- UDP-GlcNAc: dolichyl-P NAcGlc N-glycosylated proteinglycosylation CDG Ij (608093) phosphotransferase Lysosomal storageCongenital disorders of N- β-1,4-Mannosyltransferase N-glycosylatedprotein glycosylation CDG Ik (608540) Lysosomal storage Congenitaldisorders of N- α-1,2-Mannosyltransferase N-glycosylated proteinglycosylation CDG Il (608776) Lysosomal storage Congenital disorders ofN- α-1,2-Mannosyltransferase N-glycosylated protein glycosylation, typeI (pre-Golgi glycosylation defects) Lysosomal storage CystinosisCystinosin (lysosomal cystine transporter) Cysteine Lysosomal storageFabry's disease (301500) Trihexosylceramide α-galactosidaseglobotriaosylceramide Lysosomal storage Farber's disease Ceramidaselipids (lipogranulomatosis; 228000) Lysosomal storage Fucosidosis(230000) α-L-Fucosidase fucose and complex sugars Lysosomal storageGalactosialidosis (Goldberg's Protective protein/cathepsin A (PPCA)lysosomal content syndrome, combined neuraminidase and β- galactosidasedeficiency; 256540) Lysosomal storage Gaucher's disease Glucosylceramideβ-glucosidase sphingolipids Lysosomal storage Glutamylribose-5-phosphate ADP-ribose protein hydrolase glutamyl ribose5-phosphate storage disease (305920) Lysosomal storage Glycogen storagedisease type 2 alpha glucosidase glycogen (Pompe's disease) Lysosomalstorage GM1 gangliosidosis, generalized Ganglioside β-galactosidaseacidic lipid material, gangliosides Lysosomal storage GM2 activatorprotein deficiency GM2 activator protein gangliosides (Tay-Sachs diseaseAB variant, GM2A; 272750) Lysosomal storage GM2 gangliosidosisGanglioside β-galactosidase gangliosides Lysosomal storage Infantilesialic acid storage Na phosphate cotransporter, sialin sialic aciddisorder (269920) Lysosomal storage Krabbe's disease (245200)Galactosylceramide β-galactosidase sphingolipids Lysosomal storageLysosomal acid lipase deficiency Lysosomal acid lipase cholesteryl(278000) esters and triglycerides Lysosomal storage Metachromaticleukodystrophy Arylsulfatase A sulfatides (250100) Lysosomal storageMucolipidosis ML II (I-cell disease; N-Acetylglucosaminyl-1- N-linkedglycoproteins 252500) phosphotransfeerase catalytic subunit Lysosomalstorage Mucolipidosis ML III (pseudo- N-acetylglucosaminyl-1- N-linkedglycoproteins Hurler's polydystrophy) phosphotransfeerase Lysosomalstorage Mucolipidosis ML III (pseudo- Catalytic subunit N-linkedglycoproteins Hurler's polydystrophy) Type III-A (252600) Lysosomalstorage Mucolipidosis ML III (pseudo- Substrate-recognition subunitN-linked glycoproteins Hurler's polydystrophy) Type III-C (252605)Lysosomal storage Mucopolysaccharidosis MPS I H/S α-I-Iduronidaseglycosaminoglycans (Hurler-Scheie syndrome; 607015) Lysosomal storageMucopolysaccharidosis MPS I-H α-I-Iduronidase glycosaminoglycans(Hurler's syndrome; 607014) Lysosomal storage Mucopolysaccharidosis MPSII Iduronate sulfate sulfatase glycosaminoglycans (Hunter's syndrome;309900) Lysosomal storage Mucopolysaccharidosis MPS IIIHeparan-S-sulfate sulfamidase glycosaminoglycans (Sanfilippo's syndrome)Type III-A (252900) Lysosomal storage Mucopolysaccharidosis MPS IIIN-acetyl-D-glucosaminidase glycosaminoglycans (Sanfilippo's syndrome)Type III-B (252920) Lysosomal storage Mucopolysaccharidosis MPS IIIAcetyl-CoA-glucosaminide N- glycosaminoglycans (Sanfilippo's syndrome)Type III-C acetyltransferase (252930) Lysosomal storageMucopolysaccharidosis MPS III N-acetyl-glucosaminine-6-sulfate sulfataseglycosaminoglycans (Sanfilippo's syndrome) Type III-D (252940) Lysosomalstorage Mucopolysaccharidosis MPS I-S α-I-Iduronidase glycosaminoglycans(Scheie's syndrome; 607016) Lysosomal storage Mucopolysaccharidosis MPSIV Galactosamine-6-sulfate sulfatase glycosaminoglycans (Morquio'ssyndrome) Type IV-A (253000) Lysosomal storage Mucopolysaccharidosis MPSIV β-Galactosidase glycosaminoglycans (Morquio's syndrome) Type IV-B(253010) Lysosomal storage Mucopolysaccharidosis MPS IX Hyaluronidasedeficiency glycosaminoglycans (hyaluronidase deficiency; 601492)Lysosomal storage Mucopolysaccharidosis MPS VI N-Acetyl galactosamineα-4-sulfate glycosaminoglycans (Maroteaux-Lamy syndrome; sulfatase(arylsulfatase B) 253200) Lysosomal storage Mucopolysaccharidosis MPSVII β-Glucuronidase glycosaminoglycans (Sly's syndrome; 253220)Lysosomal storage Mucosulfatidosis (multiple Sulfatase-modifyingfactor-1 sulfatides sulfatase deficiency; 272200) Lysosomal storageNiemann-Pick disease type A Sphingomyelinase sphingomyelin Lysosomalstorage Niemann-Pick disease type B Sphingomyelinase sphingomyelinLysosomal storage Niemann-Pick disease Type NPC1 protein sphingomyelinC1/Type D ((257220) Lysosomal storage Niemann-Pick disease Type C2Epididymal secretory protein 1 (HE1; NPC2 sphingomyelin (607625)protein) Lysosomal storage Prosaposin deficiency (176801) Prosaposinsphingolipids Lysosomal storage Pycnodysostosis (265800) Cathepsin Kkinins Lysosomal storage Sandhoff's disease; 268800 β-Hexosaminidase Bgangliosides Lysosomal storage Saposin B deficiency (sulfatide Saposin Bsphingolipids activator deficiency) Lysosomal storage Saposin Cdeficiency (Gaucher's Saposin C sphingolipids activator deficiency)Lysosomal storage Schindler's disease Type I N-Acetyl-galactosaminidaseglycoproteins (infantile severe form; 609241) Lysosomal storageSchindler's disease Type II N-Acetyl-galactosaminidase glycoproteins(Kanzaki disease, adult-onset form; 609242) Lysosomal storageSchindler's disease Type III N-Acetyl-galactosaminidase glycoproteins(intermediate form; 609241) Lysosomal storage Sialidosis (256550)Neuraminidase 1 (sialidase) mucopolysaccharides and mucolipids Lysosomalstorage Sialuria Finnish type (Salla disease; Na phosphatecotransporter, sialin sialic acid 604369) Lysosomal storage SialuriaFrench type (269921) UDP-N-acetylglucosamine-2-epimerase/N- sialic acidacetylmannosamine kinase, sialin Lysosomal storage Sphingolipidosis TypeI (230500) Ganglioside β-galactosidase sphingolipids Lysosomal storageSphingolipidosis Type II (juvenile Ganglioside β-galactosidasesphingolipids type; 230600) Lysosomal storage Sphingolipidosis Type III(adult Ganglioside β-galactosidase sphingolipids type; 230650) Lysosomalstorage Tay-Sachs disease; 272800 β-Hexosaminidase A gangliosidesLysosomal storage Winchester syndrome (277950) Metalloproteinase-2mucopolysaccharides Lysosomal storage Wolman's disease lysosomal acidlipase lipids and cholesterol Lysosomal storage α-Mannosidosis (248500),type I α-D-Mannosidase carbohydrates and (severe) or II (mild)glycoproteins Lysosomal storage β-Mannosidosis (248510) β-D-Mannosidasecarbohydrates and glycoproteins Toxic Molecule alpha hemolysin poisoningan antibody-like binder to alpha hemolysin alpha hemolysin ToxicMolecule antrax toxin poisoning an antibody-like binder to anthrax toxinanthrax toxin Toxic Molecule bacterial toxin-induced shock anantibody-like binder to bacterial toxin bacterial toxin Toxic Moleculebotulinum toxin poisoning an antibody-like binder to botulinum toxinbotulinum toxin Toxic Molecule Hemochromatosis (iron iron chelatormolecular iron poisoning) Toxic Molecule Methanol poisoning Methanoldehdrogenase Methanol Toxic Molecule Nerve gas poisoning Butyrylcholinesterase Sarin Toxic Molecule Prion disease caused by PRP anantibody-like binder to prion protein Prion protein PRP PRP ToxicMolecule Prion disease caused by PRPc an antibody-like binder to prionprotein Prion protein PRPc PRPc Toxic Molecule Prion disease caused byPRPsc an antibody-like binder to prion protein Prion protein PRPsc PRPscToxic Molecule Prion disease cuased by PRPres an antibody-like binder toprion protein Prion protein PRPres PRPres Toxic Molecule Sepsis orcytokine storm an antibody-like binder to cytokines or cytokines Duffyantigen receptor of chemokines (DARC) Toxic Molecule spider venompoisoning an antibody-like binder to spider venom spider venom ToxicMolecule Wilson disease copper chelator molecular copper

TABLE 8 Complement & Complement Regulatory Molecules Soluble moleculesAlternative Pathway Factor B Factor D Properdin C3 C3a C3b iC3b C3c C3dgC3dk C3e Bb Factor I Classical Pathway C1q C1r C1s C4 C4a C4b C2 C4bpLectin Pathway Mannose-Binding Lectin (MBL) MBL-Associated SerineProtease 1 (MASP1) MBL-Associated Serine Protease 2 (MASP2) LateComponents C5 C5a C6 C7 C8 C9 Receptors CR1 CR2 CR3 CR4 C3aR C3eRDecay-accelerating factor (DAF) Membrane cofactor protein (MCP) CD59 C3Beta chain Receptor Homologous restriction factor Control Proteins C1inhibitor C4 binding protein Factor I Factor H

What is claimed is:
 1. A method of making an enucleated erythroid cellcomprising an exogenous antigen, the method comprising: subjecting anenucleated erythroid cell to a controlled cell injury in the presence ofthe exogenous antigen; thereby making the enucleated erythroid cellcomprising an exogenous antigen.
 2. The method of claim 1, wherein thecontrolled cell injury caused a perturbation in the cell membrane of theenucleated erythroid cell.
 3. The method of claim 1, wherein thecontrolled cell injury comprises electroporation, sonoporation,liposomal transfection, or salt-based transfection.
 4. The method ofclaim 1, wherein the controlled cell injury comprises cell deformation.5. The method of claim 1, wherein the controlled cell injury comprisescell squeezing.
 6. The method of claim 1, wherein the controlled cellinjury comprises passing the enucleated erythroid cell through amicro-channel.
 7. The method of claim 6, which comprising moving thecell through the micro-channel by application of pressure.
 8. The methodof claim 1, wherein the controlled cell injury results in the cellmembrane becoming porous.
 9. The method of claim 1, which furthercomprising isolating an enucleated erythroid cell from a subject beforesubjecting the enucleated erythroid cell to a controlled cell injury.10. The method of claim 1, further comprising autologous administrationof the enucleated erythroid cell comprising the exogenous antigen to thesubject.
 11. The method of claim 1, wherein the exogenous antigen isintracellular.
 12. The method of claim 1, wherein the exogenous antigenis present in the cytoplasm of the enucleated erythroid cell.
 13. Themethod of claim 1, wherein the enucleated erythroid cell is not ahypotonically dialysed cell.
 14. The method of claim 1, wherein apharmaceutical composition comprising a plurality of the enucleatederythroid cell comprising an exogenous antigen is capable of inducingimmune tolerance in a human subject suffering from or at risk ofdeveloping an autoimmune disease, disorder or condition.
 15. The methodof claim 1, wherein the enucleated erythroid cell comprises at least1,000 copies of the exogenous antigen.
 16. The method of claim 1,wherein the exogenous antigen is selected from insulin, proinsulin,preproinsulin, GAD65, IA-2, myelin oligodendrocyte glycoprotein, myelinbasic protein, and proteolipid protein, or an antigenic fragmentthereof.
 17. The method of claim 1, wherein the enucleated erythroidcell is a mature erythrocyte.
 18. The method of claim 1, wherein theenucleated erythroid cell is a reticulocyte.
 19. The method of claim 1,which comprises a population of erythroid cells that is greater than 80%enucleated.
 20. The method of claim 1, wherein the enucleated erythroidcells have a viability that is greater than 70%.
 21. The method of claim1, wherein at least 80% of cells in the composition comprise theexogenous antigen and are enucleated.